Fc region-containing polypeptides that exhibit improved effector function due to alterations of the extent of fucosylation, and methods for their use

ABSTRACT

The present invention relates to Fc region-containing polypeptides that exhibit improved effector function due to alterations of the extent of fucosylation, and to methods of using such polypeptides for treating or preventing cancer and other diseases. The Fc region-containing polypeptides of the present invention are preferably immunoglobulins (e.g., antibodies), in which the Fc region comprises at least one amino acid substitution relative to the corresponding amino acid sequence of a wild type Fc region, and which is sufficient to attenuate post-translational fucosylation and mediate improved binding to an activating Fc receptor and reduced binding to an inhibitory Fc receptor. The methods of the invention are particularly useful in preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection where either an enhanced efficacy of effector cell function mediated by FcγR is desired (e.g., cancer, infectious disease) or an inhibited effector cell response mediated by FcγR is desired (e.g., inflammation, autoimmune disease).

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a national application of International ApplicationNo. PCT/US2010/051831 (filed on Oct. 7, 2010), which claims priority toU.S. Patent Application Ser. No. 61/249,510 (filed on Oct. 7, 2009),which applications are herein incorporated by reference in theirentireties. This Application additionally incorporates by reference intheir entireties U.S. patent application Ser. No. 11/952,568 (filed onDec. 7, 2007) and 60/869,254 (filed on Dec. 6, 2006).

2. FIELD OF THE INVENTION

The present invention relates to Fc region-containing polypeptides thatexhibit improved effector function due to alterations of the extent offucosylation, and to methods of using such polypeptides for treating orpreventing cancer and other diseases. The Fc region-containingpolypeptides of the present invention are preferably immunoglobulins(e.g., antibodies), in which the Fc region comprises at least one aminoacid substitution relative to the corresponding amino acid sequence of awild type Fc region, and which is sufficient to attenuatepost-translational fucosylation and mediate improved binding to anactivating Fc receptor and reduced binding to an inhibitory Fc receptor.

The methods of the invention are particularly useful in preventing,treating, or ameliorating one or more symptoms associated with adisease, disorder, or infection where an enhanced efficacy of effectorcell function (e.g., ADCC) mediated by FcγR is desired, e.g. cancer,infectious disease. The methods of the invention are also of use inenhancing the therapeutic efficacy of therapeutic antibodies the effectof which is mediated by ADCC. Conversely, the methods of the inventionare particularly useful in preventing, treating, or ameliorating one ormore symptoms associated with a disease or disorder in which decreasedefficacy of effector cell function mediated by FcγR is desired, e.g.,inflammation, etc. The methods of the invention are thus also of use inenhancing the therapeutic efficacy of therapeutic antibodies whichattenuate inflammatory processes.

3. BACKGROUND OF THE INVENTION

3.1 Fc Receptors and their Roles in the Immune System

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cytotoxicity, mast celldegranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to specialized cell surface receptors onhematopoietic cells. The diversity of cellular responses triggered byantibodies and immune complexes results from the structuralheterogeneity of Fc receptors. Fc receptors share structurally relatedligand binding domains which presumably mediate intracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe α chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FεR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. 1991,Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes andInfection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal ofClinical Investigation, 2(109): 161-1681; Ravetch J. V. et al. 2000,Science, 290: 84-89; Ravetch J. V. et al., 2001 Annu. Rev. Immunol.19:275-90; Ravetch J. V. 1994, Cell, 78(4): 553-60). The different Fcreceptors, the cells that express them, and their isotype specificity iswell known in the art, see, e.g., Immunobiology: The Immune System inHealth and Disease, 4^(th) ed. 1999, Elsevier Science Ltd/GarlandPublishing, New York, which is hereby incorporated by reference in itsentirety.

Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologybetween the three family members suggest they arose from a commonprogenitor perhaps by gene duplication.

FcγRII(CD32)

FcγRII proteins are 40 KDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogenous responses to receptorligation. The fundamental difference is that the A isoform initiatesintracellular signaling leading to cell activation such as phagocytosisand respiratory burst, whereas the B isoform initiates inhibitorysignals, e.g., inhibiting B-cell activation.

Signaling through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When colligatedalong with an activating FcγR, the ITIM in FcγRIIB becomesphosphorylated and attracts the SH2 domain of the inositol polyphosphate5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengersreleased as a consequence of ITAM-containing FcγR-mediated tyrosinekinase activation, consequently preventing the influx of intracellularCa⁺⁺. Thus crosslinking of FcγRIIB dampens the activating response toFcγR ligation and inhibits cellular responsiveness. B cell activation, Bcell proliferation and antibody secretion is thus aborted.

3.2 Diseases of Relevance

3.2.1 Cancer

A neoplasm, or tumor, is a neoplastic mass resulting from abnormaluncontrolled cell growth which can be benign or malignant. Benign tumorsgenerally remain localized. Malignant tumors are collectively termedcancers. The term “malignant” generally means that the tumor can invadeand destroy neighboring body structures and spread to distant sites tocause death (for review, see Robbins and Angell, 1976, Basic Pathology,2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Cancer can arisein many sites of the body and behave differently depending upon itsorigin. Cancerous cells destroy the part of the body in which theyoriginate and then spread to other part(s) of the body where they startnew growth and cause more destruction.

More than 1.2 million Americans develop cancer each year. Cancer is thesecond leading case of death in the United States and if current trendscontinue, cancer is expected to be the leading cause of the death by theyear 2010. Lung and prostate cancer are the top cancer killers for menin the United States. Lung and breast cancer are the top cancer killersfor women in the United States. One in two men in the United States willbe diagnosed with cancer at some time during his lifetime. One in threewomen in the United States will be diagnosed with cancer at some timeduring her lifetime.

A cure for cancer has yet to be found. Current treatment options, suchas surgery, chemotherapy and radiation treatment, are oftentimes eitherineffective or present serious side effects.

Cancer Therapy

Currently, cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (See, for example, Stockdale, 1998, “Principles of CancerPatient Management”, in Scientific American: Medicine, vol. 3,Rubenstein and Federman, eds., Chapter 12, Section IV). Recently, cancertherapy could also involve biological therapy or immunotherapy. All ofthese approaches pose significant drawbacks for the patient. Surgery,for example, may be contraindicated due to the health of the patient ormay be unacceptable to the patient. Additionally, surgery may notcompletely remove the neoplastic tissue. Radiation therapy is onlyeffective when the neoplastic tissue exhibits a higher sensitivity toradiation than normal tissue, and radiation therapy can also oftenelicit serious side effects. Hormonal therapy is rarely given as asingle agent and although can be effective, is often used to prevent ordelay recurrence of cancer after other treatments have removed themajority of the cancer cells. Biological therapies/immunotherapies arelimited in number and may produce side effects such as rashes orswellings, flu-like symptoms, including fever, chills and fatigue,digestive tract problems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A significant majority ofcancer chemotherapeutics act by inhibiting DNA synthesis, eitherdirectly, or indirectly by inhibiting the biosynthesis of thedeoxyribonucleotide triphosphate precursors, to prevent DNA replicationand concomitant cell division (See, for example, Gilman et al., Goodmanand Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed.(Pergamom Press, New York, 1990)). These agents, which includealkylating agents, such as nitrosourea, anti-metabolites, such asmethotrexate and hydroxyurea, and other agents, such as etoposides,campathecins, bleomycin, doxorubicin, daunorubicin, etc., although notnecessarily cell cycle specific, kill cells during S phase because oftheir effect on DNA replication. Other agents, specifically colchicineand the vinca alkaloids, such as vinblastine and vincristine, interferewith microtubule assembly resulting in mitotic arrest. Chemotherapyprotocols generally involve administration of a combination ofchemotherapeutic agents to increase the efficacy of treatment.

Despite the availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks (See, for example, Stockdale, 1998,“Principles Of Cancer Patient Management” in Scientific AmericanMedicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10).Almost all chemotherapeutic agents are toxic, and chemotherapy causessignificant, and often dangerous, side effects, including severe nausea,bone marrow depression, immunosuppression, etc. Additionally, even withadministration of combinations of chemotherapeutic agents, many tumorcells are resistant or develop resistance to the chemotherapeuticagents. In fact, those cells resistant to the particularchemotherapeutic agents used in the treatment protocol often prove to beresistant to other drugs, even those agents that act by mechanismsdifferent from the mechanisms of action of the drugs used in thespecific treatment; this phenomenon is termed pleiotropic drug ormultidrug resistance. Thus, because of drug resistance, many cancersprove refractory to standard chemotherapeutic treatment protocols.

There is a significant need for alternative cancer treatments,particularly for treatment of cancer that has proved refractory tostandard cancer treatments, such as surgery, radiation therapy,chemotherapy, and hormonal therapy. A promising alternative isimmunotherapy, in which cancer cells are specifically targeted by cancerantigen-specific antibodies. Major efforts have been directed atharnessing the specificity of the immune response, for example,hybridoma technology has enabled the development of tumor selectivemonoclonal antibodies (See Green M. C. et al., 2000 Cancer Treat Rev.,26: 269-286; Weiner L M, 1999 Semin Oncol. 26(suppl. 14):43-51), and inthe past few years, the Food and Drug Administration has approved thefirst MAbs for cancer therapy: Rituxin (anti-CD20) for non-Hodgkin'sLymphoma and Herceptin [anti-(c-erb-2/HER-2)] for metastatic breastcancer (Suzanne A. Eccles, 2001, Breast Cancer Res., 3: 86-90). However,the potency of antibody effector function, e.g., to mediate antibodydependent cellular cytotoxicity (“ADCC”) is an obstacle to suchtreatment. Methods to improve the efficacy of such immunotherapy arethus needed.

3.2.2 Inflammatory Diseases and Autoimmune Diseases

Inflammation is a process by which the body's white blood cells andchemicals protect our bodies from infection by foreign substances, suchas bacteria and viruses. It is usually characterized by pain, swelling,warmth and redness of the affected area. Chemicals known as cytokinesand prostaglandins control this process, and are released in an orderedand self-limiting cascade into the blood or affected tissues. Thisrelease of chemicals increases the blood flow to the area of injury orinfection, and may result in the redness and warmth. Some of thechemicals cause a leak of fluid into the tissues, resulting in swelling.This protective process may stimulate nerves and cause pain. Thesechanges, when occurring for a limited period in the relevant area, workto the benefit of the body.

In autoimmune and/or inflammatory disorders, the immune system triggersan inflammatory response when there are no foreign substances to fightand the body's normally protective immune system causes damage to itsown tissues by mistakenly attacking self. There are many differentautoimmune disorders which affect the body in different ways. Forexample, the brain is affected in individuals with multiple sclerosis,the gut is affected in individuals with Crohn's disease, and thesynovium, bone and cartilage of various joints are affected inindividuals with rheumatoid arthritis. As autoimmune disorders progressdestruction of one or more types of body tissues, abnormal growth of anorgan, or changes in organ function may result. The autoimmune disordermay affect only one organ or tissue type or may affect multiple organsand tissues. Organs and tissues commonly affected by autoimmunedisorders include red blood cells, blood vessels, connective tissues,endocrine glands (e.g., the thyroid or pancreas), muscles, joints, andskin. Examples of autoimmune disorders include, but are not limited to,Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, dermatomyositis, lupuserythematosus, multiple sclerosis, autoimmune inner ear diseasemyasthenia gravis. Reiter's syndrome, Graves disease, autoimmunehepatitis, familial adenomatous polyposis and ulcerative colitis.

Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are types ofinflammatory arthritis. Arthritis is a general term that describesinflammation in joints. Some, but not all, types of arthritis are theresult of misdirected inflammation. Besides rheumatoid arthritis, othertypes of arthritis associated with inflammation include the following:psoriatic arthritis, Reiter's syndrome, ankylosing spondylitisarthritis, and gouty arthritis. Rheumatoid arthritis is a type ofchronic arthritis that occurs in joints on both sides of the body (suchas both hands, wrists or knees). This symmetry helps distinguishrheumatoid arthritis from other types of arthritis. In addition toaffecting the joints, rheumatoid arthritis may occasionally affect theskin, eyes, lungs, heart, blood or nerves.

Rheumatoid arthritis affects about 1% of the world's population and ispotentially disabling. There are approximately 2.9 million incidences ofrheumatoid arthritis in the United States. Two to three times more womenare affected than men. The typical age that rheumatoid arthritis occursis between 25 and 50. Juvenile rheumatoid arthritis affects 71,000 youngAmericans (aged eighteen and under), affecting six times as many girlsas boys.

Rheumatoid arthritis is an autoimmune disorder where the body's immunesystem improperly identifies the synovial membranes that secrete thelubricating fluid in the joints as foreign. Inflammation results, andthe cartilage and tissues in and around the joints are damaged ordestroyed. In severe cases, this inflammation extends to other jointtissues and surrounding cartilage, where it may erode or destroy boneand cartilage and lead to joint deformities. The body replaces damagedtissue with scar tissue, causing the normal spaces within the joints tobecome narrow and the bones to fuse together. Rheumatoid arthritiscreates stiffness, swelling, fatigue, anemia, weight loss, fever, andoften, crippling pain. Some common symptoms of rheumatoid arthritisinclude joint stiffness upon awakening that lasts an hour or longer;swelling in a specific finger or wrist joints; swelling in the softtissue around the joints; and swelling on both sides of the joint.Swelling can occur with or without pain, and can worsen progressively orremain the same for years before progressing.

The diagnosis of rheumatoid arthritis is based on a combination offactors, including: the specific location and symmetry of painfuljoints, the presence of joint stiffness in the morning, the presence ofbumps and nodules under the skin (rheumatoid nodules), results of X-raytests that suggest rheumatoid arthritis, and/or positive results of ablood test called the rheumatoid factor. Many, but not all, people withrheumatoid arthritis have the rheumatoid-factor antibody in their blood.The rheumatoid factor may be present in people who do not haverheumatoid arthritis. Other diseases can also cause the rheumatoidfactor to be produced in the blood. That is why the diagnosis ofrheumatoid arthritis is based on a combination of several factors andnot just the presence of the rheumatoid factor in the blood.

The typical course of the disease is one of persistent but fluctuatingjoint symptoms, and after about 10 years, 90% of sufferers will showstructural damage to bone and cartilage. A small percentage will have ashort illness that clears up completely, and another small percentagewill have very severe disease with many joint deformities, andoccasionally other manifestations of the disease. The inflammatoryprocess causes erosion or destruction of bone and cartilage in thejoints. In rheumatoid arthritis, there is an autoimmune cycle ofpersistent antigen presentation, T-cell stimulation, cytokine secretion,synovial cell activation, and joint destruction. The disease has a majorimpact on both the individual and society, causing significant pain,impaired function and disability, as well as costing millions of dollarsin healthcare expenses and lost wages. (See, for example, the NIHwebsite and the NIAID website).

Currently available therapy for arthritis focuses on reducinginflammation of the joints with anti-inflammatory or immunosuppressivemedications. The first line of treatment of any arthritis is usuallyanti-inflammatories, such as aspirin, ibuprofen and Cox-2 inhibitorssuch as celecoxib and rofecoxib. “Second line drugs” include gold,methotrexate and steroids. Although these are well-establishedtreatments for arthritis, very few patients remit on these lines oftreatment alone. Recent advances in the understanding of thepathogenesis of rheumatoid arthritis have led to the use of methotrexatein combination with antibodies to cytokines or recombinant solublereceptors. For example, recombinant soluble receptors for tumor necrosisfactor (TNF)-α have been used in combination with methotrexate in thetreatment of arthritis. However, only about 50% of the patients treatedwith a combination of methotrexate and anti-TNF-α agents such asrecombinant soluble receptors for TNF-α show clinically significantimprovement. Many patients remain refractory despite treatment.Difficult treatment issues still remain for patients with rheumatoidarthritis. Many current treatments have a high incidence of side effectsor cannot completely prevent disease progression. So far, no treatmentis ideal, and there is no cure. Novel therapeutics are needed that moreeffectively treat rheumatoid arthritis and other autoimmune disorders.

3.2.3 Infectious Diseases

Infectious agents that cause disease fall into five groups: viruses,bacteria, fungi, protozoa, and helminths (worms). The remarkable varietyof these pathogens has caused the natural selection of two crucialfeatures of adaptive immunity. First, the advantage of being able torecognize a wide range of different pathogens has driven the developmentof receptors on B and T cells of equal or greater diversity. Second, thedistinct habitats and life cycles of pathogens have to be countered by arange of distinct effector mechanisms. The characteristic features ofeach pathogen are its mode of transmission, its mechanism ofreplication, its pathogenesis or the means by which it causes disease,and the response it elicits.

The record of human suffering and death caused by smallpox, cholera,typhus, dysentery, malaria, etc. establishes the eminence of theinfectious diseases. Despite the outstanding successes in controlafforded by improved sanitation, immunization, and antimicrobialtherapy, the infectious diseases continue to be a common and significantproblem of modern medicine. The most common disease of mankind, thecommon cold, is an infectious disease, as is the feared modern diseaseAIDS. Some chronic neurological diseases that were thought formerly tobe degenerative diseases have proven to be infectious. There is littledoubt that the future will continue to reveal the infectious diseases asmajor medical problems.

An enormous number of human and animal diseases result from virulent andopportunistic infections from any of the above mentioned infectiousagents (see Belshe (Ed.) 1984 Textbook of Human Virology, PSGPublishing, Littleton, Mass.).

One category of infectious diseases are viral infections for example.Viral diseases of a wide array of tissues, including the respiratorytract, CNS, skin, genitourinary tract, eyes, ears, immune system,gastrointestinal tract, and musculoskeletal system, affect a vast numberof humans of all ages (see Table 328-2 In: Wyngaarden and Smith, 1988,Cecil Textbook of Medicine, 18^(th) Ed, W.B. Saunders Co., Philadelphia,pp. 1750-1753). Although considerable effort has been invested in thedesign of effective anti-viral therapies, viral infections continue tothreaten the lives of millions of people worldwide. In general, attemptsto develop anti-viral drugs have focused on several stages of viral lifecycle (See e.g., Mitsuya et al., 1991, FASEB J. 5:2369-2381, discussingHIV). However, a common drawback associated with using of many currentanti-viral drugs is their deleterious side effects, such as toxicity tothe host or resistance by certain viral strains.

4. SUMMARY OF THE INVENTION

The invention relates to methods of treating or preventing cancer andother diseases, disorders and infections using molecules, preferablypolypeptides, and more preferably immunoglobulins (e.g., antibodies),comprising a variant Fc region, having one or more amino acidmodifications (e.g., substitutions, but also including insertions ordeletions) in one or more regions, which modification(s) alter (relativeto a wild-type Fc region) the Ratio of Affinities of the variant Fcregion to an activating FcγR (such as FcγRIIA or FcγRIIIA) relative toan inhibiting FcγR (such as FcγRIIB):

${{Ratio}\mspace{14mu}{of}\mspace{14mu}{Affinities}} = \frac{{Wild}\text{-}{Type}{\mspace{11mu}\;}{to}\mspace{14mu}{Variant}\mspace{14mu}{Change}\mspace{14mu}{in}\mspace{14mu}{Affinity}\mspace{14mu}{to}\mspace{14mu}{Fc}\;\gamma\; R_{Activating}}{{Wild}\text{-}{Type}\mspace{14mu}{to}\mspace{14mu}{Variant}\mspace{14mu}{Change}\mspace{14mu}{in}\mspace{14mu}{Affinity}\mspace{14mu}{to}\mspace{14mu}{Fc}\;\gamma\; R_{Inhibiting}}$

Of particular interest are Ratios of Affinities in which either FcγRIIIAor FcγRIIA is the FcγR_(Activating) and FcγRIIB is theFcγR_(Inhibiting). Where an Fc variant has a Ratio of Affinities greaterthan 1, the methods of the invention have particular use in providing atherapeutic or prophylactic treatment of a disease, disorder, orinfection, or the amelioration of a symptom thereof, where an enhancedefficacy of effector cell function (e.g., ADCC) mediated by FcγR isdesired, e.g., cancer or infectious disease. Such an increased Ratio ofAffinities may result from the Fc region of the molecule having(relative to a wild type Fc) an increase in affinity to anFcγR_(Activating) (e.g., FcγRIIIA or FcγRIIA) coupled with either anunchanged affinity to an FcγR_(Inhibiting) (e.g., FcγRIIB) or a decreasein affinity to such FcγR_(Inhibiting). Alternatively, an increased Ratioof Affinities may result from the Fc region of such molecule exhibitingan increase in affinity to both an FcγR_(Activating) and anFcγR_(Inhibiting) (relative to a wild-type Fc), provided that theincrease in affinity to the FcγR_(Activating) exceeds the increase inaffinity to the FcγR_(Inhibiting), or may result from the Fc region ofsuch molecule exhibiting a decreased affinity to both theFcγR_(Activating) and an FcγR_(Inhibiting) (relative to a wild-type Fc),provided that the decrease in affinity to the FcγR_(Activating) is lessthan the decrease in affinity to the FcγR_(Inhibiting), or may resultfrom an unchanged affinity to an FCγR_(Activating) coupled with adecrease in affinity to an FcγR_(Inhibiting).

Where an Fv variant has a Ratio of Affinities less than 1, the methodsof the invention have particular use in providing a therapeutic orprophylactic treatment of a disease or disorder, or the amelioration ofa symptom thereof, where a decreased efficacy of effector cell functionmediated by FcγR is desired, e.g., autoimmune or inflammatory disorders.Such a decreased Ratio of Affinities may result from the Fc region ofthe molecule having (relative to a wild type Fc) a decrease in affinityto an FcγR_(Activating) (e.g., FcγRIIIA or FcγRIIA) coupled with eitheran unchanged affinity to an FcγR_(Inhibiting) (e.g., FcγRIIB) or anincrease in affinity to such FcγR_(Inhibiting). Alternatively, adecreased Ratio of Affinities may result from the Fc region of suchmolecule exhibiting a decrease in affinity to both an FcγR_(Activating)and an FcγR_(Inhibiting) (relative to a wild-type Fc), provided that thedecrease in affinity to the FcγR_(Activating) exceeds the decrease inaffinity to the FcγR_(Inhibiting), or may result from the Fc region ofsuch molecule exhibiting an increased affinity to both anFcγR_(Activating) and an FcγR_(Inhibiting) (relative to a wild-type Fc),provided that the increase in affinity to the FcγR_(Activating) is lessthan the increase in affinity to the FcγR_(Inhibiting), or may resultfrom an unchanged affinity to an FcγR_(Activating) coupled with anincrease in affinity to an FCγR_(Inhibiting).

Current approaches to optimize the Fc region function (e.g.,antibody-dependent cell mediated cytotoxicity (ADCC), complementdependent cytotoxicity (CDC) activity) in therapeutic monoclonalantibodies and soluble polypeptides fused to Fc regions have focused ona limited number of single amino acid changes based on structuralanalysis and/or computer aided designs. Alternative approaches inengineering Fc regions have focused on the glycosylation of the Fcregion to optimize Fc region function. The validity of using an Fcvariant's Ratio of Affinities to assess its therapeutic potential hasbeen suggested with respect to Fc variants whose sequences were derivedusing computer algorithms to search sequence-structure space (Lazar, G.A. et al. Proc. Natl. Acad. Sci. (USA) 103:4005-4010 (2006)). Thisapproach identified four Fc variants: (1) S239D; (2) 1322E; (3) S239Dand I322E; and (4) S239D, 1332E and A330L, all of which bound FcγRIIIaas well as FcγRIIb with greater affinity than wild-type (Lazar. G. A. etal. Proc. Natl. Acad. Sci. (USA) 103:4005-4010 (2006). In contrast, thepresent invention is based, in part, on selecting desired variantFc-containing molecules that exhibit an altered Ratio of Affinities forFcγRIII and FcγRII, from an unbiased library of Fc variants. Thisapproach enabled the identification of a larger universe of desired Fcvariants, as well as variants having Ratios of Affinities far in excessof those reported by Lazar, G. A. et al. (Proc. Natl. Acad. Sci. (USA)103:4005-4010 (2006)). The present invention encompasses methods forengineering Fc regions and identification and screening of novel Fcvariants outside the expected regions identified by structural studies.Expected regions as used herein refer to those regions that based onstructural and/or biochemical studies are in contact with an Fc ligand.

The therapeutic or prophylactic molecules that are used in accordancewith the methods of the invention thus comprise variant Fc regionscomprising one or more amino acid modifications that exhibit an alteredRatio of Affinities, especially wherein the FcγR_(Activating) is eitherFcγRIIA or FcγRIIIA and the FcγR_(Inhibiting) is FcγRIIB. In a preferredembodiment, the molecules of the invention further specifically bindFcγRIIB (via the Fc region) with a lower affinity than a comparablemolecule (i.e., having the same amino acid sequence as the molecule ofthe invention except for the one or more amino acid modifications in theFc region) comprising the wild-type Fc region binds FcγRIIB. In someembodiments, the invention encompasses molecules with variant Fcregions, having one or more amino acid modifications, whichmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA and enhance the affinity of the variant Fcregion for FcγRIIB relative to a comparable molecule with a wild type Fcregion. In other embodiments, the invention encompasses molecules withvariant Fc regions, having one or more amino acid modifications, whichmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA but do not alter the affinity of the variant Fcregions for FcγRIIB relative to a comparable molecule with a wild typeFc region. A preferred embodiment is a variant Fc region that hasenhanced affinity for FcγRIIIA and FcγRIIA but reduced affinity forFcγRIIB relative to a comparable molecule with a wild type Fc region.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that altereffector function. The invention encompasses combining an Fc variant ofthe invention with other Fc modifications to provide additive,synergistic, or novel properties in antibodies or Fc fusions.Preferably, the Fc variants of the invention enhance the phenotype ofthe modification with which they are combined. For example, if an Fcvariant of the invention is combined with a mutant known to bindFcγRIIIA with a higher affinity than a comparable molecule comprising awild type Fc region; the combination with a mutant of the inventionresults in a greater fold enhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:49634969;Armour et al, 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, JImmunol 164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Shieldset al, 2002, J Biol Chem 277:26733-26740; Jefferis et al, 2002, ImmunolLett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490);Lazar, G. A. et al. Proc. Natl. Acad. Sci. (USA) 103:4005-4010 (2006);U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573; U.S. Pat. No.6,194,551; PCT WO 00/42072; PCT WO 99/58572; PCT WO 04/063351; U.S.Patent Application Publication 2005/0037000; and U.S. Patent ApplicationPublication 2005/0064514; each of which is incorporated herein byreference in its entirety. In certain embodiments, the Fc variants ofthe present invention may be combined with one or more of the Fcvariants, i.e., amino acid modifications relative to a wild-type Fcregion, presented in tables 4, 5, 9, and 10, infra.

The invention encompasses molecules that are homodimers or heterodimersof Fc regions. Heterodimers comprising Fc regions refer to moleculeswhere the two Fc chains have the same or different sequences. In someembodiments, in the heterodimeric molecules comprising variant Fcregions, each chain has one or more different modifications from theother chain. In other embodiments, in the heterodimeric moleculescomprising variant Fc regions, one chain contains the wild-type Fcregion and the other chain comprises one or more modifications. Methodsof engineering heterodimeric Fc containing molecules are known in theart and encompassed within the invention.

In some embodiments, the invention encompasses molecules comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region binds an FcγR_(Activating) but does not bind anFcγR_(Inhibiting) or binds an FcγR_(Inhibiting) with a reduced affinity,relative to a comparable molecule comprising the wild-type Fc region, asdetermined by standard assays (e.g., in vitro assays) known to oneskilled in the art. In an alternative embodiment, the inventionencompasses molecules comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild type Fc region, which variant Fc region binds anFcγR_(Inhibiting), does not bind an FcγR_(Activating) or binds anFcγR_(Activating) with reduced affinity, relative to a comparablemolecule comprising the wild-type Fc region, as determined by standardassays (e.g., in vitro assays) known to one skilled in the art. In aspecific embodiment, the invention encompasses molecules comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region only binds one FcγR, wherein said FcγR is FcγIIIA. In anotherspecific embodiment, the invention encompasses molecules comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region only binds one FcγR, wherein said FcγR is FcγRIIA. In yetanother embodiment, the invention encompasses molecules comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region only binds one FcγR, wherein said FcγR is FcγRIIB.

The affinities and binding properties of the molecules of the inventionfor an FcγR are initially determined using in vitro assays (biochemicalor immunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to ELISA assay, surface plasmon resonanceassay, immunoprecipitation assays (See Section 6.2). Preferably, thebinding properties of the molecules of the invention are alsocharacterized by in vitro functional assays for determining one or moreFcγR mediator effector cell functions (See Section 6.2.2). In mostpreferred embodiments, the molecules of the invention have similarbinding properties in in vivo models (such as those described anddisclosed herein) as those in in vitro based assays. However, thepresent invention does not exclude molecules of the invention that donot exhibit the desired phenotype in in vitro based assays but doexhibit the desired phenotype in vivo.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc regionand exhibits a Ratio of Affinities greater than 1, provided that saidvariant Fc region does not solely have a substitution at any one ofpositions 329, 331, or 332, or does not include, or is not solely asubstitution of, any one of: (1) alanine at any of positions 256, 290,298, 312, 333, 334, 359, 360, 326, or 430; (2) a lysine at position 330;(3) a threonine at position 339; (4) a methionine at position 320; (5) aserine at position 326; (6) an asparagine at position 326; (7) anaspartic acid at position 326; (8) a glutamic acid at position 326; (9)a glutamine at position 334; (10) a glutamic acid at position 334; (11)a methionine at position 334; (12) a histidine at position 334; (13) avaline at position 334; (14) a leucine at position 334; (15) a lysine atposition 335, or (16) solely a glutamic acid at position 332; (17)solely a glutamic acid at position 332 and an aspartic acid at position239; (18) solely a glutamic acid at position 332, an aspartic acid atposition 239, and a leucine at position 330.

The invention particularly concerns a molecule having such a variant Fcregion, wherein said variant Fc region is additionally characterized inpossessing at least one amino acid modification relative to a wild-typeFc region at position 234, 235, 243, 247, 255, 270, 284, 292, 300, 305,316, 370, 392, 396, 416, 419 and/or 421.

The invention further concerns such molecules wherein the variant Fcregions are additionally characterized in possessing substitutions at atleast the two positions: (a) 235 and 243; (b) 243 and 292; (c) 243 and300; (d) 243 and 305; (e) 243 and 396; (f) 247 and 270; (g) 247 and 421;(h) 255 and 270; (i) 255 and 396; (j) 270 and 316; (k) 270 and 396; (l)270 and 416; (m) 270 and 421; (n) 292 and 300; (o) 292 and 305; (p) 292and 396; (q) 300 and 396; (r) 305 and 396; (s) 316 and 416; (t) 392 and270; (u) 392 and 396; (v) 419 and 270; or (w) 419 and 396.

The invention further concerns such molecules wherein the variant Fcregions are additionally characterized in possessing substitutions at atleast the three positions: ((a) 243, 247 and 421; (b) 243, 292 and 300;(c) 243, 292 and 305; (d) 243, 292 and 396; (e) 243 300 and 396; (f)243, 305 and 396; (g) 247, 270 and 421; (h) 255, 270 and 396; (i) 270,316 and 416; (j) 270, 392 and 396; (k) 270, 396 and 419; (l) 292 300 and396; or (m) 292, 305 and 396.

The invention further concerns the embodiments of such antibodieswherein the at least one modification in the Fc domain comprises asubstitution of L234 or a substitution of L235, or substitutions of bothL234 and L235, and particularly, wherein the substitution of L234 isL234F and said substitution at L235 is L235V.

The invention additionally concerns the above-described methods whereinthe variant Fc region possesses at least amino acid modificationsrelative to a wild-type Fc region at one or more of positions: 243, 292,300 or 396 (i.e., at position 243, 292, 300 or 396; or at both position243 and at position 292; or at both position 243 and at position 300; orat both position 243 and at position 396; or at both position 292 and atposition 300; or at both position 292 and at position 396; or at bothposition 300 and at position 396; or at position 243, at position 292and at position 300; or at position 243, at position 292 and at position396; or at position 292, at position 300 and at position 396; or atposition 243, at position 292, at position 300 and at position 396). Theinvention particularly concerns the embodiment of the above-describedmethods wherein such variant Fc region possess the modifications: L234F,L235I, F243L, R292P and/or Y300L.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities greater than 1, and wherein suchvariant Fc regions have at least any of the following substitutions: (a)F243L; (b) D270E; (c) R292G; or (d) R292P.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities greater than 1, and wherein suchvariant Fc regions have at least any of the following pairs ofsubstitutions: (a) F243L and R292P; (b) F243L and Y300L; (c) F243L andP396L; (d) D270E and P396L; (e) R292P and Y300L; (f) R292P and V3051;(g) R292P and P396L; (h) Y300L and P396L; and (i) P396L and Q419H.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities greater than 1, and wherein suchvariant Fc regions have at least any of the following trios ofsubstitutions: (a) F243L, P247L and N421K; (b) F243L, R292P and Y300L;(c) F243L, R292P and Y300L; (d) F243L, R292P and V305I; (e) F243L, R292Pand P396L; (f) F243L, Y300L and P396L; (g) P247L, D270E and N421K; (h)R255L, D270E and P396L; (i) D270E, G316D and R416G; (j) D270E, K392T andP396L; (k) D270E, P396L and Q419H; (l) V284M, R292L and K370N or (m)R292P, Y300L and P396L.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities greater than 1, and wherein suchvariant Fc regions have at least any of the following tetrads ofsubstitutions: (a) L234F, F243L, R292P and Y300L; (b) L235I, F243L,R292P and Y300L; (c) L235Q, F243L, R292P and Y300L; (d) F243L, R292P,Y300L, and P396L; (e) F243L, P247L, D270E and N421K; (f) F243L, R255L,D270E and P396L; (g) F243L, D270E, G316D and R416G; (h) F243L, D270E,K392T and P396L; (i) F243L, D270E, P396L and Q419H; (j) F243L, R292P,V305I and P396L; (k) D270E, G316D, P396L and R416G; (l) P247L, D270E,Y300L and N421K; (m) R255L, D270E, R292G and P396L; or (n) R255L, D270E,Y300L and P396L.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities greater than 1, and wherein suchvariant Fc regions have at least any of the following pentads ofsubstitutions: (a) L235V, F243L, R292P, Y300L and P396L; (b) L235P,F243L, R292P, Y300L and P396L; (c) F243L, R292P, V305I, Y300L and P396L;or (d) F243L, R292P, Y300L, V305I and P396L.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities less than 1, and wherein suchvariant Fc regions have at least any of the following substitutions: (a)P396L or (b) Y300L.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities less than 1, and wherein suchvariant Fc regions have at least any of the following pairs ofsubstitutions: (a) F243L and P396L; (b) P247L and N421K; (c) R255L andP396L; (d) R292P and V305I; (e) K392T and P396L; or (f) P396L and Q419H.

The invention further concerns such molecules wherein the variant Fcregions exhibit a Ratio of Affinities less than 1, and wherein suchvariant Fc regions have at least the following three substitutions:F243L, R292P and V3051.

In another specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIA with a greateraffinity than a comparable molecule comprising the wild-type Fc regionbinds FcγRIIA, provided that the one or more amino acid modifications donot include or are not solely substitution with an alanine at any ofpositions 256, 290, 326, 255, 258, 267, 272, 276, 280, 283, 285, 286,331, 337, 268, 272, or 430; an asparagine at position 268; a glutamineat position 272; a glutamine, serine, or aspartic acid at position 286;a serine at position 290; a methionine, glutamine, glutamic acid, orarginine at position 320; a glutamic acid at position 322; a serine,glutamic acid, or aspartic acid at position 326; a lysine at position330; a glutamine at position 335; or a methionine at position 301.

In a preferred specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule has an altered affinity for an FcγR, providedthat said variant Fc region does not have a substitution at positionsthat make a direct contact with FcγR based on crystallographic andstructural analysis of Fc-FcγR interactions such as those disclosed bySondermann et al., (2000 Nature, 406: 267-273, which is incorporatedherein by reference in its entirety). Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(hinge region), amino acids 265-269 (B/C loop), amino acids 297-299(C′/E loop), and amino acids 327-332 (F/G) loop. In some embodiments,the molecules of the invention comprising variant Fc regions comprisemodification of at least one residue that does not make a direct contactwith an FcγR based on structural and crystallographic analysis, e.g., isnot within the Fc-FcγR binding site.

In another preferred embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule binds an FcγR (via its Fc region) with analtered affinity relative to a molecule comprising a wild-type Fcregion, provided that said at least one amino acid modification do notinclude or are not solely a substitution at any of positions 255, 256,258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290,292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320,322, 326, 329, 330, 332, 331, 333, 334, 335, 337, 338, 339, 340, 359,360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculebinds an FcγR (via its Fc region) with an altered affinity relative to amolecule comprising a wild-type Fc region, provided that said variant Fcregion does not include or are not solely a substitution at any ofpositions 255, 258, 267, 269, 270, 276, 278, 280, 283, 285, 289, 292,293, 294, 295, 296, 300, 303, 305, 307, 309, 322, 329, 332, 331, 337,338, 340, 373, 376, 416, 419, 434, 435, 437, 438, 439 and does not havean alanine at any of positions 256, 290, 298, 312, 333, 334, 359, 360,326, or 430; a lysine at position 330: a threonine at position 339; amethionine at position 320; a serine at position 326; an asparagine atposition 326; an aspartic acid at position 326; a glutamic acid atposition 326; a glutamine at position 334; a glutamic acid at position334; a methionine at position 334; a histidine at position 334; a valineat position 334; or a leucine at position 334; a lysine at position 335an asparagine at position 268; a glutamine at position 272; a glutamine,serine, or aspartic acid at position 286; a serine at position 290; amethionine, glutamine, glutamic acid, or arginine at position 320; aglutamic acid at position 322; a serine, glutamic acid, or aspartic acidat position 326; a lysine at position 330; a glutamine at position 335;or a methionine at position 301.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region does notinclude or are not solely a substitution at any of positions 268, 269,270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307,309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430,434, 435, 437, 438 or 439 and does not have a histidine, glutamine, ortyrosine at position 280; a serine, glycine, threonine or tyrosine atposition 290, a leucine or isoleucine at position 300; an asparagine atposition 294, a proline at position 296; a proline, asparagine, asparticacid, or valine at position 298; a lysine at position 295. In yetanother preferred embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule binds an FcγR (via its Fc region) with a reducedaffinity relative to a molecule comprising a wild-type Fc regionprovided that said variant Fc region does not have or are not solelyhave a substitution at any of positions 252, 254, 265, 268, 269, 270,278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327,329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434,435, 437, 438, or 439. In yet another preferred embodiment, theinvention encompasses a molecule comprising a variant Fc region, whereinsaid variant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said molecule binds an FcγR(via its Fc region) with an enhanced affinity relative to a moleculecomprising a wild-type Fc region provided that said variant Fc regiondoes not have or are not solely a substitution at any of positions 280,283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315,331, 333, 334, 337, 340, 360, 378, 398, or 430.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region does notinclude a substitution or does not solely have a substitution at any ofpositions 330, 243, 247, 298, 241, 240, 244, 263, 262, 235, 269, or 328and does not have a leucine at position 243, an asparagine at position298, a leucine at position 241, and isoleucine or an alanine at position240, a histidine at position 244, a valine at position 330, or anisoleucine at position 328.

In a specific embodiment, molecules of the invention comprise a variantFc region having one or more amino acid modifications (e.g.,substitutions), which modifications increase the affinity of the variantFc region for FcγRIIIA and/or FcγRIIA by at least 2-fold, relative to acomparable molecule comprising a wild-type Fc region. In certainembodiments, molecules of the invention comprise a variant Fc regionhaving one or more amino acid modifications (e.g., substitutions), whichmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA by greater than 2-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 8-fold, or at least 10-foldrelative to a comparable molecule comprising a wild-type Fc region. Inother embodiments of the invention, molecules of the inventioncomprising a variant Fc region specifically bind FcγRIIIA and/or FcγRIIAwith at least 65%, at least 75%, at least 85%, at least 95%, at least100%, at least 150%, at least 200% greater affinity relative to amolecule comprising a wild-type Fc region. Such measurements arepreferably in vitro assays.

The invention encompasses molecules with altered affinities for theactivating and/or inhibitory Fcγ receptors. In particular, the inventioncontemplates molecules with variant Fc regions, having one or more aminoacid modifications, which modifications increase the affinity of thevariant Fc region for FcγRIIB but decrease the affinity of the variantFc region for FcγRIIIA and/or FcγRIIA, relative to a comparable moleculewith a wild-type Fc region. In other embodiments, the inventionencompasses molecules with variant Fc regions, having one or more aminoacid modifications, which modifications decrease the affinity of thevariant Fc region for FcγRIIB and also decrease the affinity of thevariant Fc regions for FcγRIIIA and/or FcγRIIA relative to a comparablemolecule with a wild-type Fc region. In yet other embodiments, theinvention encompasses molecules with variant Fc regions, having one ormore amino acid modifications, which modifications increase the affinityof the variant Fc region for FcγRIIB and also increase the affinity ofthe variant Fc regions for FcγRIIIA and/or FcγRIIA relative to acomparable molecule with a wild-type Fc region. In yet otherembodiments, the invention encompasses molecules with variant Fcregions, which modifications decrease the affinity of the variant Fcregion for FcγRIIIA and/or FcγRIIA but do not alter the affinity of thevariant Fc region for FcγRIIB relative to a comparable molecule with awild-type Fc region. In yet other embodiments, the invention encompassesmolecules with variant Fc regions, which modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA but reducethe affinity of the variant Fc region for FcγRIIB relative to acomparable molecule with a wild-type Fc region.

In a specific embodiment, the molecules of the invention comprise avariant Fc region, having one or more amino acid modifications (e.g.,substitutions), which one or more modifications increase the affinity ofthe variant Fc region for FcγRIIIA and decrease the affinity of thevariant Fc region for FcγRIIB, relative to a comparable moleculecomprising a wild-type Fc region which binds FcγRIIIA and FcγRIIB withwild-type affinity. In a certain embodiment, the one or more amino acidmodifications are not a substitution with alanine at any of positions256, 298, 333, or 334.

In another specific embodiment, the molecules of the invention comprisea variant Fc region, having one or more amino acid modifications (e.g.,substitutions), which one or more modifications increase the affinity ofthe variant Fc region for FcγRIIA and decrease the affinity of thevariant Fc region for FcγRIIB, relative to a comparable moleculecomprising a wild-type Fc region which binds FcγRIIA and FcγRIIB withwild-type affinity. In a certain embodiment, the one or more amino acidmodifications is not a substitution with arginine at position 320.

In most preferred embodiments, the molecules of the invention withaltered affinities for activating and/or inhibitory receptors havingvariant Fc regions, have one or more amino acid modifications, whereinsaid one or more amino acid modification is a substitution at position288 with asparagine, at position 330 with serine and at position 396with leucine (MgFc10); or a substitution at position 334 with glutamicacid, at position 359 with asparagine, and at position 366 with serine(MgFc13); or a substitution at position 316 with aspartic acid, atposition 378 with valine, and at position 399 with glutamic acid(MgFc27); or a substitution at position 392 with threonine, and atposition 396 with leucine (MgFc38); or a substitution at position 221with glutamic acid, at position 270 with glutamic acid, at position 308with alanine, at position 311 with histidine, at position 396 withleucine, and at position 402 with aspartic acid (MgFc42); or asubstitution at position 240 with alanine, and at position 396 withleucine (MgFc52); or a substitution at position 410 with histidine, andat position 396 with leucine (MgFc53); or a substitution at position 243with leucine, at position 305 with isoleucine, at position 378 withaspartic acid, at position 404 with serine, and at position 396 withleucine (MgFc54); or a substitution at position 255 with isoleucine, andat position 396 with leucine (MgFc55); or a substitution at position 370with glutamic acid and at position 396 with leucine (MgFc59); or asubstitution at position 243 with leucine, at position 292 with proline,at position 300 with leucine, at position 305 with isoleucine, and atposition 396 with leucine (MgFc88); or a substitution at position 243with leucine, at position 292 with proline, at position 300 withleucine, and at position 396 with leucine (MgFc88A); or a substitutionat position 243 with leucine, at position 292 with proline, and atposition 300 with leucine (MgFc155); or a substitution at position 243with leucine, at position 292 with proline, and at position 300 withleucine; or a substitution at position 243 with leucine, at position 292with proline, and at position 396 with leucine; or a substitution atposition 243 with leucine, and at position 292 with proline; or asubstitution at position 243 with leucine; or a substitution at position273 with phenylalanine; or a substitution at position 247 with leucine,at position 270 with glutamic acid, and at position 421 with lysine. Ina related embodiment, the variant Fc region further comprises one ormore amino acid modifications disclosed in tables 4, 5, 9, and 10 infra.

In certain embodiments, the invention encompasses methods for screeningand identifying therapeutic and/or prophylactic molecules comprisingvariant Fc regions with altered FcγR affinities (e.g., enhanced FcγRIIIAaffinity) using yeast surface display technology (for review see Boderand Wittrup, 2000, Methods in Enzymology, 328: 430-444, which isincorporated herein by reference in its entirety). Yeast surface displayof the mutant Fc containing polypeptides of the invention may beperformed in accordance with any of the techniques known to thoseskilled in the art or described herein (see, e.g., U.S Patentapplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351, each of whichis hereby incorporated by reference in its entirety). Yeast displayoffers the advantage of utilizing actual binding to a desired receptorto identify variant Fc regions that have enhanced binding to thatreceptor.

In certain embodiments, the invention encompasses methods for screeningand identifying therapeutic and/or prophylactic molecules comprisingvariant Fc regions with altered FcγR affinities (e.g., enhanced FcγRIIIAaffinity) using yeast display technology known in the art or describedherein in combination with one or more biochemical based assays,preferably in a high throughput manner. The one or more biochemicalassays can be any assay known in the art for identifying Fc-FcγRinteraction, i.e., specific binding of an Fc region to an FcγR,including, but not limited to, an ELISA assay, surface plasmon resonanceassays, immunoprecipitation assay, affinity chromatography, andequilibrium dialysis. In some embodiments, screening and identifyingmolecules comprising variant Fc regions with altered FcγR affinities(e.g., enhanced FcγRIIIA affinity) are done using the yeast displaytechnology as described herein in combination with one or morefunctional based assays, preferably in a high throughput manner. Thefunctional based assays can be any assay known in the art forcharacterizing one or more FcγR mediated effector cell function such asthose described herein in Section 6.2.2. Non-limiting examples ofeffector cell functions that can be used in accordance with the methodsof the invention, include but are not limited to, antibody-dependentcell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis,phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting,C1q binding, and complement dependent cell mediated cytotoxicity. Insome embodiments, screening and identifying molecules comprising variantFc regions with altered FcγR affinities (e.g., enhanced FcγRIIIAaffinity) are done using the yeast display technology as describedherein or known in the art in combination with one or more biochemicalbased assays in combination or in parallel with one or more functionalbased assays, preferably in a high throughput manner.

In preferred embodiments, the invention encompasses methods forscreening and characterizing FcγR-Fc interaction using biochemicalassays developed by the inventors and disclosed in U.S Patentapplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351, each of whichis hereby incorporated by reference in its entirety. The disclosedassays allow detection and quantitation of the FcγR-Fc interaction,despite the inherently weak affinity of the receptor for its ligand,e.g., in the micromolar range for FcγRIIB and FcγRIIIA. The methodinvolves the formation of an FcγR complex (e.g., FcγRIIIA, FcγRIIB) thathas an improved avidity for an Fc region, relative to an uncomplexedFcγR.

The invention encompasses the use of the immune complexes formedaccording to the methods described above for determining thefunctionality of molecules comprising an Fc region in cell-based orcell-free assays.

In preferred embodiments, molecules of the invention (e.g.,immunoglobulins or fragments thereof) comprising the variant Fc regionsare further characterized in an animal model for interaction with anFcγR or in an animal model of disease state. Preferred animal models foruse in the methods of the invention are, for example, transgenic miceexpressing human FcγRs, e.g., any mouse model described in U.S. Pat.Nos. 5,877,397, and 6,676,927 which are incorporated herein by referencein their entirety. Transgenic mice for use in the methods of theinvention include, but are not limited to, knockout FcγRIIIA micecarrying human FcγRIIIA; knockout FcγRIIIA mice carrying human FcγRIIA;knockout FcγRIIIA mice carrying human FcγRIIB and human FcγRIIIA;knockout FcγRIIIA mice carrying human FcγRIIB and human FcγRIIA;knockout FcγRIIIA and FcγRIIA mice carrying human FcγRIIIA and FcγRIIA;and knockout FcγRIIIA, FcγRIIA and FcγRIIB mice carrying human FcγRIIIA,FcγRIIA and FcγRIIB. The mouse strain used for knockout studies may beany suitable inbred strain (e.g., B6) as determined routinely in theart. In preferred embodiments, the mouse strain is that of a nudegenotype, i.e., immune compromised, to allow xenograft studies (e.g.,cancer models). Such nude strains include, but are not limited to FoxN1and N/N. In other embodiments the mice carrying one or more human FcγRsfurther comprise one or more additional genetic mutations including oneor more knockouts, e.g. RAG1−/−.

In a specific embodiment, the invention provides modifiedimmunoglobulins comprising a variant Fc region with an enhanced affinityfor FcγRIIIA and/or FcγRIIA. Such immunoglobulins include IgG moleculesthat naturally contain FcγR binding regions (e.g., FcγRIIIA and/orFcγRIIB binding regions), or immunoglobulin derivatives that have beenengineered to contain an FcγR binding region (e.g., FcγRIIIA and/orFcγRIIB binding regions). The modified immunoglobulins of the inventioninclude any immunoglobulin molecule that binds, preferably,immunospecifically, i.e., competes off non-specific binding asdetermined by immunoassays well known in the art for assaying specificantigen-antibody binding, an antigen and contains an FcγR binding region(e.g., a FcγRIIIA and/or FcγRIIB binding region). Such antibodiesinclude, but are not limited to, polyclonal, monoclonal, bi-specific,multi-specific, human, humanized, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs, andfragments containing either a VL or VH domain or even a complementarydetermining region (CDR) that specifically binds an antigen, in certaincases, engineered to contain or fused to an FcγR binding region.

In certain embodiment, the invention encompasses immunoglobulinscomprising a variant Fc region with an enhanced affinity for FcγRIIIAand/or FcγRIIA such that the immunoglobulin has an enhanced effectorfunction, e.g., antibody dependent cell mediated cytotoxicity. Theeffector function of the molecules of the invention can be assayed usingany assay described herein or known to those skilled in the art. In someembodiments, immunoglobulins comprising a variant Fc region with anenhanced affinity for FcγRIIIA and/or FcγRIIA have an enhanced ADCCactivity relative to wild-type by at least 2-fold, at least 4-fold, atleast 8-fold, at least 10-fold, at least 50-fold, or at least 100-fold.

The invention encompasses engineering human or humanized therapeuticantibodies (e.g., tumor specific monoclonal antibodies) in the Fc regionby modification (e.g., substitution, insertion, deletion) of one or moreamino acid residues, which modifications modulate the affinity of thetherapeutic antibody for an FcγR activating receptor and/or an FcγRinhibitory receptor. In one embodiment, the invention relates toengineering human or humanized therapeutic antibodies (e.g., tumorspecific monoclonal antibodies) in the Fc region by modification of oneor more amino acid residues, which modifications increase the affinityof the Fc region for FcγRIIIA and/or FcγRIIA. In another embodiment, theinvention relates to engineering human or humanized therapeuticantibodies (e.g., tumor specific monoclonal antibodies) in the Fc regionby modification of one or more amino acid residues, which modificationincreases the affinity of the Fc region for FcγRIIIA and/or FcγRIIA andfurther decreases the affinity of the Fc region for FcγRIIB. Theengineered therapeutic antibodies may further have an enhanced effectorfunction, e.g., enhanced ADCC activity, phagocytosis activity, etc., asdetermined by standard assays known to those skilled in the art.

In a specific embodiment, the invention encompasses engineering amonoclonal antibody specific for Her2/neu protooncogene (amino acidsequence SEQ ID NO:31) (e.g., 4D5 antibody as disclosed in Carter etal., 1992, Proc. Natl. Acad. Sci. USA 89:4285-9; U.S. Pat. No.5,677,171; or International Patent Application Publication WO 01/00245,each of which is hereby incorporated by references in its entirety) bymodification (e.g., substitution, insertion, deletion) of at least oneamino acid residue which modification increases the affinity of the Fcregion for FcγRIIIA and/or FcγRIIA. In another specific embodiment,modification of the humanized Her2/neu monoclonal antibody may alsofurther decrease the affinity of the Fc region for FcγRIIB. In yetanother specific embodiment, the engineered humanized monoclonalantibodies specific for Her2/neu may further have an enhanced effectorfunction as determined by standard assays known in the art and disclosedand exemplified herein. In a certain embodiment, the 4D5 antibody ischimeric. In another embodiment, the 4D5 antibody is humanized. In aspecific embodiment, the 4D5 antibody to be engineered in accordancewith the methods of the invention comprises a heavy chain having theamino acid sequence SEQ ID NO:32. In another specific embodiment, the4D5 antibody to be engineered in accordance with the methods of theinvention comprises a light chain having the amino acid sequence SEQ IDNO:33. In still other embodiments, the 4D5 antibody to be engineered inaccordance with the methods of the invention is humanized and comprisesa heavy chain having the amino sequence SEQ ID NO:34. In furtherembodiments, the 4D5 antibody to engineered in accordance with themethods of the invention is humanized and comprises a light chain havingthe amino sequence SEQ ID NO:35.

In a specific embodiment, the antibodies of the invention bind Her2/neu.The anti-Her2/neu antibodies of the invention may have a heavy chainvariable region comprising the amino acid sequence of CDR1 (SEQ IDNO:36) and/or CDR2 (SEQ ID NO:37) and/or CDR3 (SEQ ID NO:38) and/or alight chain variable region comprising the amino acid sequence of CDR1(SEQ ID NO:39) and/or a CDR2 (SEQ ID NO:40) and/or CDR3 (SEQ ID NO:41).

In a specific embodiment, the invention encompasses a 4D5 antibody(e.g., chimeric, humanized) comprising a variant Fc region having asubstitution at position 243 with leucine, at position 292 with proline,at position 300 with leucine, at position 305 with isoleucine, and atposition 396 with leucine. In another specific embodiment, the inventionencompasses a 4D5 antibody having a leucine at position 243, a prolineat position 292, a leucine at position 300, an isoleucine at position305, and a leucine at position 396. In other embodiments, the inventionencompasses a 4D5 antibody comprising a variant Fc region having asubstitution at position 243 with leucine, at position 292 with proline,and at position 300 with leucine. In other embodiments, the inventionencompasses a 4D5 antibody having a leucine at position 243, a prolineat position 292, and a leucine at position 300. In other embodiments,the invention encompasses a 4D5 antibody comprising a variant Fc regionhaving a substitution at position 247 with leucine, at position 270 withglutamic acid, and at position 421 with lysine. In another embodiment,the invention encompasses a 4D5 antibody having a leucine at position247, a glutamic acid at position 292, and a lysine at position 421.

In another specific embodiment, the invention encompasses engineering ananti-CD20 antibody by modification (e.g., substitution, insertion,deletion) of at least one amino acid residue which modificationincreases the affinity of the Fc region for FcγRIIIA and/or FcγRIIA. Ina related embodiment, the anti-CD20 antibody is mouse human chimericanti-CD20 monoclonal antibody, 2H7Further nonlimiting examples ofanti-CD20 antibodies that can be used in the methods of the inventionare disclosed in U.S. patent application Ser. No. 11/271,140, filed Nov.10, 2005, hereby incorporated by reference in its entirety. In anotherspecific embodiment, modification of the anti-CD20 monoclonal antibody,2H7 may also further decrease the affinity of the Fc region for FcγRIIB.In yet another specific embodiment, the engineered anti-CD20 monoclonalantibody, 2H7 may further have an enhanced effector function asdetermined by standard assays known in the art and disclosed andexemplified herein.

In a specific embodiment, the invention encompasses a 2H7 antibody(e.g., chimeric, humanized) comprising a variant Fc region having asubstitution at position 243 with leucine, at position 292 with proline,at position 300 with leucine, at position 305 with isoleucine, and atposition 396 with leucine. In another specific embodiment, the inventionencompasses a 2H7 antibody having a leucine at position 243, a prolineat position 292, a leucine at position 300, an isoleucine at position305, and a leucine at position 396. In other embodiments, the inventionencompasses a 4D5 antibody comprising a variant Fc region having asubstitution at position 243 with leucine, at position 292 with proline,and at position 300 with leucine. In other embodiments, the inventionencompasses a 2H7 antibody having a leucine at position 243, a prolineat position 292, and a leucine at position 300. In other embodiments,the invention encompasses a 2H7 antibody comprising a variant Fc regionhaving a substitution at position 247 with leucine, at position 270 withglutamic acid, and at position 421 with lysine. In another embodiment,the invention encompasses a 2H7 antibody having a leucine at position247, a glutamic acid at position 292, and a lysine at position 421.

In another specific embodiment, the invention encompasses engineering ananti-FcγRIIB antibody, in particular an anti-FcγRIIB antibody thatspecifically binds human FcγRIIB, more particularly native humanFcγRIIB, by modification (e.g. substitution, insertion, deletion) of atleast one amino acid residue which modification increases the affinityof the Fc region for FcγRIIIA and/or FcγRIIA. Non-limiting examples ofrepresentative anti-FcγRIIB antibodies are disclosed in U.S. ProvisionalApplication No. 60/403,266 filed on Aug. 12, 2002; U.S. application Ser.No. 10/643,857 filed on Aug. 14, 2003; and U.S Patent ApplicationPublication Numbers: 2004-0185045; 2005-0260213; and 2006-0013810, allof which are hereby incorporated by reference in their entireties.Examples of anti-FcγRIIB antibodies that may be engineered in accordancewith the methods of the invention are the monoclonal antibodies producedby clone 2B6, 3H7, 8B5.4.3, 1D5, 2E1, 2H9, 2D11, 8B5 and 1F2 having ATCCaccession numbers PTA-4591, PTA-4592, PTA-7610, PTA-5958, PTA-5961,PTA-5962, PTA-5960, PTA-7610 and PTA-5959, respectively (deposited atATCC, 10801 University Boulevard, Manassas, Va. 02209-2011, all of whichare incorporated herein by reference), or chimeric, humanized or otherengineered versions thereof.

In a specific embodiment, the invention encompasses engineering ahumanized antibody comprising the heavy chain variable domain and/orlight chain variable domain of 2B6, 3H7 or 8B5.3.4. In another specificembodiment, the invention encompasses engineering a humanized antibodycomprising the CDRs of 2B6, 3H7 or 8B5.3.4. In a specific embodiment,the invention encompasses engineering a humanized antibody comprisingthe heavy chain variable domain having the amino acid sequence of SEQ IDNO: 1, SEQ ID NO:2 or SEQ ID NO:3 and the light chain variable domainhaving the amino acid sequence of SEQ ID NO: 4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7 or SEQ ID NO: 8. In a specific embodiment, theinvention encompasses engineering a humanized antibody comprising theheavy chain variable domain having the amino acid sequence of SEQ IDNO:9 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:10.

In a specific embodiment, the invention encompasses engineering ahumanized 2B6 antibody comprising a heavy chain having the amino acidsequence SEQ ID NO:42. In another specific embodiment, the inventionencompasses engineering a humanized 2B6 antibody comprising a heavychain having the amino acid sequence SEQ ID NO:29. In still otherembodiments, the invention encompasses engineering a humanized 2B6antibody comprising a light chain having the amino acid sequence SEQ IDNO:30. In a preferred embodiment, the invention encompasses engineeringa humanized 2B6 antibody comprising a heavy chain containing the aminoacid sequence SEQ ID NO:29 and a light chain containing the sequence SEQID NO:30. In a specific aspect of the invention, the inventionencompasses the use of plasmid pMGx0675, which includes the nucleotidesequences SEQ ID NO:43 and SEQ ID NO:44 that encode the heavy chainamino acid sequence SEQ ID NO:29 and the light chain amino acid sequenceSEQ ID NO:30, respectively. Plasmid pMGx0675 been deposited with theAmerican Type Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on May 23, 2006 under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedures, and assigned accession number PTA7609, and is incorporated herein by reference.

In specific embodiments, the invention encompasses engineeringantibodies, preferably humanized, that bind the extracellular domain ofnative human FcγRIIB. The humanized anti-FcγRIIB antibodies encompassedby the invention may have a heavy chain variable region comprising theamino acid sequence of CDR1 (SEQ ID NO:15, SEQ ID NO:16, an amino acidsequence corresponding to amino acids 31-35 as set forth in SEQ ID NO:2,or an amino acid sequence corresponding to amino acids 31-35 as setforth in SEQ ID NO:3) and/or CDR2 (SEQ ID NO:17, SEQ ID NO:18, an aminoacid sequence corresponding to amino acids 50-66 as set forth in SEQ IDNO:2, or an amino acid sequence corresponding to amino acids 50-66 asset forth in SEQ ID NO:3) and/or CDR3 (SEQ ID NO:19, SEQ ID NO:20, anamino acid sequence corresponding to amino acids 100-111 as set forth inSEQ ID NO:2, or an amino acid sequence corresponding to amino acids100-111 as set forth in SEQ ID NO:3) and/or a light chain variableregion comprising the amino acid sequence of CDR1 (SEQ ID NO:21, SEQ IDNO:22, or an amino acid sequence corresponding to amino acids 24-34 asset forth in SEQ ID NO:8) and/or a CDR2 (SEQ ID NO:23, SEQ ID NO:24, SEQID NO:25, SEQ ID NO:26, or an amino acid sequence corresponding to aminoacids 50-56 as set forth in SEQ ID NO:62) and/or CDR3 (SEQ ID NO:27, SEQID NO:28, or an amino acid sequence corresponding to amino acids 90-98as set forth in SEQ ID NO:8).

In a specific embodiment, the invention encompasses a 2B6 antibodycomprising a variant Fc region having a substitution at position 243with leucine, at position 292 with proline, at position 300 withleucine, at position 305 with isoleucine, and at position 396 withleucine. In another specific embodiment, the invention encompasses a 2B6antibody having a leucine at position 243, a proline at position 292, aleucine at position 300, an isoleucine at position 305, and a leucine atposition 396. In other embodiments, the invention encompasses a 2B6antibody comprising a variant Fc region having a substitution atposition 243 with leucine, at position 292 with proline, and at position300 with leucine. In other embodiments, the invention encompasses a 2B6antibody having a leucine at position 243, a proline at position 292,and a leucine at position 300. In other embodiments, the inventionencompasses a 2B6 antibody comprising a variant Fc region having asubstitution at position 247 with leucine, at position 270 with glutamicacid, and at position 421 with lysine. In another embodiment, theinvention encompasses a 2B6 antibody having a leucine at position 247, aglutamic acid at position 292, and a lysine at position 421.

In a specific embodiment, modification of the anti-FcγRIIB antibody mayalso decrease the affinity of the Fc region for FcγRIIB relative to thewild-type antibody. In yet another specific embodiment, the engineeredanti-FcγRIIB antibody may further have an enhanced effector function asdetermined by standard assays known in the art and disclosed andexemplified herein. In a specific embodiment, the anti-FcγRIIBmonoclonal antibody comprises a modification at position 334 withglutamic acid, at position 359 with asparagine, and at position 366 withserine (MgFc13); or a substitution at position 316 with aspartic acid,at position 378 with valine, and at position 399 with glutamic acid(MgFc27); or a substitution at position 243 with isoleucine, at position379 with leucine, and at position 420 with valine (MgFc29); or asubstitution at position 392 with threonine and at position 396 withleucine (MgFc38); or a substitution at position 221 with glutamic acid,at position 270 with glutamic acid, at position 308 with alanine, atposition 311 with histidine, at position 396 with leucine, and atposition 402 with aspartic (MgFc42); or a substitution at position 410with histidine, and at position 396 with leucine (MgFc53); or asubstitution at position 243 with leucine, at position 305 withisoleucine, at position 378 with aspartic acid, at position 404 withserine, and at position 396 with leucine (MgFc54); or a substitution atposition 255 with isoleucine, and at position 396 with leucine (MgFc55);or a substitution at position 370 with glutamic acid, and at position396 with leucine (MgFc59); or a substitution at position 243 withleucine, at position 292 with proline, at position 300 with leucine, atposition 305 with isoleucine, and at position 396 with leucine (MgFc88);or a substitution at position 243 with leucine, at position 292 withproline, at position 300 with leucine, and at position 396 with leucine(MgFc88A); or a substitution at position 234 with leucine, at position292 with proline, and at position 300 with leucine (MgFc155); or asubstitution at position 243 with leucine, at position 292 with proline,and at position 300 with leucine; or a substitution at position 243 withleucine, at position 292 with proline, and at position 396 with leucine;or a substitution at position 243 with leucine, and at position 292 withproline; or a substitution at position 243 with leucine; or asubstitution at position 273 with phenylalanine; or a substitution atposition 247 with leucine, at position 270 with glutamic acid, and atposition 421 with lysine. In a related embodiment, the variant Fc regionfurther comprises one or more amino acid modifications disclosed intables 4, 5, 9, and 10, infra.

In a specific embodiments, the invention encompasses an antibody thatbinds to CD79a or CD79b (e.g., chimeric, humanized) comprising a variantFc region and use of the antibodies for treatment of cancer. In specificembodiments, the antibody that binds to CD79b or CD79b has asubstitution at position 243 with leucine, at position 292 with proline,at position 300 with leucine, at position 305 with isoleucine, and atposition 396 with leucine. In another specific embodiment, the inventionencompasses an antibody that binds to CD79a or CD79b having a leucine atposition 243, a proline at position 292, a leucine at position 300, anisoleucine at position 305, and a leucine at position 396. In otherembodiments, the invention an antibody that binds to CD79a or CD79breceptor comprising a variant Fc region having a substitution atposition 243 with leucine, at position 292 with proline, and at position300 with leucine. In other embodiments, the invention encompasses anantibody that binds to CD79a or CD79b having a leucine at position 243,a proline at position 292, and a leucine at position 300. In otherembodiments, the invention encompasses an antibody that binds to CD79aor CD79b comprising a variant Fc region having a substitution atposition 247 with leucine, at position 270 with glutamic acid, and atposition 421 with lysine. In another embodiment, the inventionencompasses an antibody that binds to CD79a or CD79b having a leucine atposition 247, a glutamic acid at position 292, and a lysine at position421.

In a specific embodiments, the invention encompasses an antibody thatbinds to ErbB1 (e.g., chimeric, humanized) comprising a variant Fcregion and use of the antibodies for treatment of cancer. In specificembodiments, the antibody that binds to ErbB1 has a substitution atposition 243 with leucine, at position 292 with proline, at position 300with leucine, at position 305 with isoleucine, and at position 396 withleucine. In another specific embodiment, the invention encompasses anantibody that binds to ErbB1 having a leucine at position 243, a prolineat position 292, a leucine at position 300, an isoleucine at position305, and a leucine at position 396. In other embodiments, the inventionan antibody that binds to ErbB1 receptor comprising a variant Fc regionhaving a substitution at position 243 with leucine, at position 292 withproline, and at position 300 with leucine. In other embodiments, theinvention encompasses an antibody that binds to ErbB1 having a leucineat position 243, a proline at position 292, and a leucine at position300. In other embodiments, the invention encompasses an antibody thatbinds to ErbB1 comprising a variant Fc region having a substitution atposition 247 with leucine, at position 270 with glutamic acid, and atposition 421 with lysine. In another embodiment, the inventionencompasses an antibody that binds to ErbB1 having a leucine at position247, a glutamic acid at position 292, and a lysine at position 421.

In a specific embodiments, the invention encompasses an antibody thatbinds to A33, CD5, CD11c, CD19, CD22, CD23, CD27, CD40, CD45, CD103,CTLA4, ErbB3, ErbB4, VEGF receptor, TNF-a receptor, TNF-β receptor, orTNF-γ receptor (e.g., chimeric, humanized) comprising a variant Fcregion and use of the antibodies for treatment of cancer. In specificembodiments, the antibody that binds to A33, CD5, CD11c, CD19, CD22,CD23, CD27, CD40, CD45, CD103, CTLA4, ErbB3, ErbB4, VEGF receptor, TNF-areceptor, TNF-β receptor, or TNF-γ receptor has a substitution atposition 243 with leucine, at position 292 with proline, at position 300with leucine, at position 305 with isoleucine, and at position 396 withleucine. In another specific embodiment, the invention encompasses anantibody that binds to A33, CD5, CD11c, CD19, CD22, CD23, CD27, CD40,CD45, CD103, CTLA4, ErbB3, ErbB4, VEGF receptor, TNF-a receptor, TNF-βreceptor, or TNF-γ receptor having a leucine at position 243, a prolineat position 292, a leucine at position 300, an isoleucine at position305, and a leucine at position 396. In other embodiments, the inventionan antibody that binds to A33, CD5, CD11c, CD19, CD22, CD23, CD27, CD40,CD45, CD103, CTLA4, ErbB3, ErbB4, VEGF receptor, TNF-a receptor, TNF-βreceptor, or TNF-γ receptor comprising a variant Fc region having asubstitution at position 243 with leucine, at position 292 with proline,and at position 300 with leucine. In other embodiments, the inventionencompasses an antibody that binds to A33, CD5, CD11c, CD19, CD22, CD23,CD27, CD40, CD45, CD103, CTLA4, ErbB3, ErbB4, VEGF receptor, TNF-areceptor, TNF-β receptor, or TNF-γ receptor having a leucine at position243, a proline at position 292, and a leucine at position 300. In otherembodiments, the invention encompasses an antibody that binds to A33,CD5, CD11c, CD19, CD22, CD23, CD27, CD40, CD45, CD103, CTLA4, ErbB3,ErbB4, VEGF receptor, TNF-a receptor, TNF-13 receptor, or TNF-γ receptorcomprising a variant Fc region having a substitution at position 247with leucine, at position 270 with glutamic acid, and at position 421with lysine. In another embodiment, the invention encompasses anantibody that binds to A33, CD5, CD11c, CD19, CD22, CD23, CD27, CD40,CD45, CD103, CTLA4, ErbB3, ErbB4, VEGF receptor, TNF-a receptor, TNF-βreceptor, or TNF-γ receptor having a leucine at position 247, a glutamicacid at position 292, and a lysine at position 421.

The present invention also includes polynucleotides that encode amolecule of the invention, including polypeptides and antibodies,identified by the methods of the invention. The polynucleotides encodingthe molecules of the invention may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart. The invention relates to an isolated nucleic acid encoding amolecule of the invention. The invention also provides a vectorcomprising said nucleic acid. The invention further provides host cellscontaining the vectors or polynucleotides of the invention.

The invention further provides methods for the production of themolecules of the invention. The molecules of the invention, includingpolypeptides and antibodies, can be produced by any method known tothose skilled in the art, in particular, by recombinant expression. In aspecific embodiment, the invention relates to a method for recombinantlyproducing a molecule of the invention, said method comprising: (i)culturing in a medium a host cell comprising a nucleic acid encodingsaid molecule, under conditions suitable for the expression of saidmolecule; and (ii) recovery of said molecule from said medium.

The molecules identified in accordance with the methods of the inventionare useful in preventing, treating, or ameliorating one or more symptomsassociated with a disease, disorder, or infection. The molecules of theinvention are particularly useful for the treatment or prevention of adisease or disorder where an enhanced efficacy of effector cell function(e.g., ADCC) mediated by FcγR is desired, e.g., cancer, infectiousdisease, and in enhancing the therapeutic efficacy of therapeuticantibodies the effect of which is mediated by ADCC.

In one embodiment, the invention encompasses a method of treating cancerin a patient having a cancer characterized by a cancer antigen, saidmethod comprising administering a therapeutically effective amount of atherapeutic antibody that binds the cancer antigen, which has beenengineered in accordance with the methods of the invention. In aspecific embodiment, the invention encompasses a method for treatingcancer in a patient having a cancer characterized by a cancer antigen,said method comprising administering a therapeutically effective amountof a therapeutic antibody that specifically binds said cancer antigen,said therapeutic antibody comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said therapeutic antibodyspecifically binds FcγRIIIA via its Fc region with a greater affinitythan the therapeutic antibody comprising the wild-type Fc region bindsFcγRIIIA, provided that said variant Fc region does not have asubstitution at positions 329, 331, or 332, and does not have an alanineat any of positions 256, 290, 298, 312, 333, 334, 359, 360, or 430; alysine at position 330; a threonine at position 339; a methionine atposition 320; a serine at position 326; an asparagine at position 326;an aspartic acid at position 326; a glutamic acid at position 326; aglutamine at position 334; a glutamic acid at position 334; a methionineat position 334; a histidine at position 334; a valine at position 334;or a leucine at position 334. In another specific embodiment, theinvention encompasses a method for treating cancer in a patient having acancer characterized by a cancer antigen, said method comprisingadministering a therapeutically effective amount of a therapeuticantibody that specifically binds a cancer antigen, said therapeuticantibody comprising a variant Fc region, wherein said variant Fc regioncomprises at least one amino acid modification relative to a wild-typeFc region such that said therapeutic antibody specifically bindsFcγRIIIA via its Fc region with a greater affinity than a therapeuticantibody comprising the wild-type Fc region binds FcγRIIIA, and saidtherapeutic antibody further specifically binds FcγRIIB with a loweraffinity than a therapeutic antibody comprising the wild-type Fc regionbinds FcγRIIB, provided that said variant Fc region does not have analanine at any of positions 256, 298, 333, or 334. The inventionencompasses a method for treating cancer in a patient characterized by acancer antigen, said method comprising administering a therapeuticallyeffective amount of a therapeutic antibody that specifically binds saidcancer antigen and said therapeutic antibody comprises a variant Fcregion so that the antibody has an enhanced ADCC activity.

The invention encompasses a method of treating an autoimmune disorderand/or inflammatory disorder in a patient in need thereof, said methodcomprising administering to said patient a therapeutically effectiveamount of a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild type Fc region, such that said molecule specificallybinds FcγRIIB via its Fc region with a greater affinity than acomparable molecule comprising the wild type Fc region, and saidmolecule further specifically binds FcγRIIIA via its Fc region with alower affinity than a comparable molecule comprising the wild type Fcregion, and said molecule binds an immune complex (e.g., anantigen/antibody complex). The invention encompasses a method oftreating an autoimmune disorder and/or inflammatory disorder furthercomprising administering one or more additional prophylactic ortherapeutic agents, e.g., immunomodulatory agents, anti-inflammatoryagents, used for the treatment and/or prevention of such diseases.

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylactically effective amount of one or moremolecules of the invention that bind an infectious agent or cellularreceptor therefor. Infectious diseases that can be treated or preventedby the molecules of the invention are caused by infectious agentsincluding but not limited to viruses, bacteria, fungi, protozae, andviruses.

According to one aspect of the invention, molecules of the inventioncomprising variant Fc regions have an enhanced antibody effectorfunction towards an infectious agent, e.g., a pathogenic protein,relative to a comparable molecule comprising a wild-type Fc region. In aspecific embodiment, molecules of the invention enhance the efficacy oftreatment of an infectious disease by enhancing phagocytosis and/oropsonization of the infectious agent causing the infectious disease. Inanother specific embodiment, molecules of the invention enhance theefficacy of treatment of an infectious disease by enhancing ADCC ofinfected cells causing the infectious disease.

In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or additional therapeutic agents known to those skilled inthe art for the treatment and/or prevention of an infectious disease.The invention contemplates the use of the molecules of the invention incombination with antibiotics known to those skilled in the art for thetreatment and or prevention of an infectious disease.

The invention provides pharmaceutical compositions comprising a moleculeof the invention, e.g., a polypeptide comprising a variant Fc region, animmunoglobulin comprising a variant Fc region, a therapeutic antibodyengineered in accordance with the invention, and a pharmaceuticallyacceptable carrier. The invention additionally provides pharmaceuticalcompositions further comprising one or more additional therapeuticagents, including but not limited to anti-cancer agents,anti-inflammatory agents, immunomodulatory agents.

4.1 Definitions

As used herein, the term “Fc region” is used to define a C-terminalregion of an IgG heavy chain. Although the boundaries may vary slightly,the human IgG heavy chain Fc region is defined to stretch from Cys226 tothe carboxy terminus. The Fc region of an IgG comprises two constantdomains, CH2 and CH3. The CH2 domain of a human IgG Fc region (alsoreferred to as “Cγ2” domain) usually extends from amino acid 231 toamino acid 338. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG.

Throughout the present specification, the numbering of the residues inan IgG heavy chain is that of the EU index as in Kabat et al., Sequencesof Proteins of Immunological Interest, 5^(th) Ed. Public Health Service,NH1, MD (1991), expressly incorporated herein by references. The “EUindex as in Kabat” refers to the numbering of the human IgG1 EUantibody.

The “hinge region” is generally defined as stretching from Glu216 toPro230 of human IgG1. Hinge regions of other IgG isotypes may be alignedwith the IgG1 sequence by placing the first and last cysteine residuesforming inter-heavy chain S—S bonds in the same positions.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the term “variant Fc region” is intended to denote an Fcregion that has been modified, by substitution, insertion or deletion ofone or more amino acid residues relative to the Fc region of theunmodified molecule (i.e., the “wild-type” immunoglobulin). The presentinvention relates to molecules that possess Fc regions having an alteredRatio of Affinities for an FcγR that activates a cellular effectorfunction (i.e., “an FcγR_(Activating),” such as FcγRIIA or FcγRIIIA)relative to an FcγR that inhibits a cellular effector function (i.e.,“an FcγR_(Inhibiting),” such as FcγRIIB):

${{Ratio}\mspace{14mu}{of}\mspace{14mu}{Affinities}} = \frac{{Wild}\text{-}{Type}{\mspace{11mu}\;}{to}\mspace{14mu}{Variant}\mspace{14mu}{Change}\mspace{14mu}{in}\mspace{14mu}{Affinity}\mspace{14mu}{to}\mspace{14mu}{Fc}\;\gamma\; R_{Activating}}{{Wild}\text{-}{Type}\mspace{14mu}{to}\mspace{14mu}{Variant}\mspace{14mu}{Change}\mspace{14mu}{in}\mspace{14mu}{Affinity}\mspace{14mu}{to}\mspace{14mu}{Fc}\;\gamma\; R_{Inhibiting}}$

Thus, for any particular molecule having a variant Fc region, themolecule's Ratio of Affinities is determined by calculating thedifference in affinity of the variant Fc region of the molecule to anFcγR_(Activating) (for example, to FcγRIIA or to FcγRIIIA), relative tothe affinity of a wild-type immunoglobulin to suchFcγR_(Activating)(e.g., Affinity_(FcγRIIA) of the variant Fcregion—Affinity_(FcγRIIIA) of the wild-type immunoglobulin), anddividing such difference by the difference in affinity to anFcγR_(Inhibiting) (for example, FcγRIIB) of the variant Fc region of themolecule, relative to the affinity of a wild-type immunoglobulin to suchFcγR_(Inhibiting) (e.g., Affinity_(FcγRIIB) of the variant Fcregion—Affinity_(FcγRIIB) of the wild-type immunoglobulin). An increasedRatio of Affinities may result from the Fc region of the molecule having(relative to a wild type Fc) an increase in affinity to anFcγR_(Activating) (for example, to FcγRIIA or to FcγRIIIA) coupled witheither an unchanged affinity to an FcγR_(Inhibiting) (for example,FcγRIIB) or a decrease in affinity to such FcγR_(Inhibiting).Alternatively, an increased Ratio of Affinities may result from the Fcregion of such molecule exhibiting an increase in affinity to both anFcγR_(Activating) and an FcγR_(Inhibiting) (relative to a wild-type Fc),provided that the increase in affinity to the FcγR_(Activating) exceedsthe increase in affinity to the FcγR_(Inhibiting) (i.e., binding to theFcγR_(Activating) is “greatly” increased compared to binding to theFcγR_(Inhibiting), which is merely increased), or may result from the Fcregion of such molecule exhibiting a decreased affinity to both anFcγR_(Activating) and an FcγR_(Inhibiting) (relative to a wild-type Fc),provided that the decrease in affinity to the FcγR_(Activating) is lessthan the decrease in affinity to an FcγR_(Inhibiting) (i.e., binding tothe FcγR_(Inhibiting) is “greatly” decreased compared to binding to theFcγR_(Activating), which is merely decreased), or may result from anunchanged affinity to an FcγR_(Activating) coupled with a decrease inaffinity to an FcγR_(Inhibiting). A decreased Ratio of Affinities mayresult from the Fc region of the molecule having (relative to a wildtype Fc) a decrease in affinity to an FcγR_(Activating) coupled witheither an unchanged affinity to an FcγR_(Inhibiting) or an increase inaffinity to an FcγR_(Inhibiting). Alternatively, a decreased Ratio ofAffinities may result from the Fc region of such molecule exhibiting adecrease in affinity to both an FcγR_(Activating) and anFcγR_(Inhibiting) (relative to a wild-type. Fc), provided that thedecrease in affinity to the FcγR_(Activating) exceeds the decrease inaffinity to the FcγR_(Inhibiting) (i.e., binding to theFcγR_(Activating) is “greatly” decreased compared to binding to theFcγR_(Inhibiting), which is merely decreased), or may result from the Fcregion of such molecule exhibiting an increased affinity to both anFcγR_(Activating) and an FcγR_(Inhibiting) (relative to a wild-type Fc),provided that the increase in affinity to the FcγR_(Activating) is lessthan the increase in affinity to the an FcγR_(Inhibiting) (i.e., bindingto the FcγR_(Inhibiting) is “greatly” increased compared to binding tothe FcγR_(Activating), which is merely increased), or may result from anunchanged affinity to an FcγR_(Activating) coupled with an increase inaffinity to an FcγR_(Inhibiting).

As used herein, the term “derivative” in the context of polypeptides orproteins refers to a polypeptide or protein that comprises an amino acidsequence which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative” asused herein also refers to a polypeptide or protein which has beenmodified, i.e., by the covalent attachment of any type of molecule tothe polypeptide or protein. For example, but not by way of limitation,an antibody may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. A derivative polypeptide or protein may beproduced by chemical modifications using techniques known to those ofskill in the art, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Further, a derivative polypeptide or protein derivative possesses asimilar or identical function as the polypeptide or protein from whichit was derived.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes leukemias and lymphomas. In some embodiments,cancer refers to a benign tumor, which has remained localized. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Insome embodiments, the cancer is associated with a specific cancerantigen.

As used herein, the term “immunomodulatory agent” and variations thereofrefer to an agent that modulates a host's immune system. In certainembodiments, an immunomodulatory agent is an immunosuppressant agent. Incertain other embodiments, an immunomodulatory agent is animmunostimulatory agent. Immunomodatory agents include, but are notlimited to, small molecules, peptides, polypeptides, fusion proteins,antibodies, inorganic molecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to a fragment of a polypeptideor protein or a non-protein molecule having antigenic or immunogenicactivity in an animal, preferably in a mammal, and most preferably in ahuman. An epitope having immunogenic activity is a fragment of apolypeptide or protein that elicits an antibody response in an animal.An epitope having antigenic activity is a fragment of a polypeptide orprotein to which an antibody immunospecifically binds as determined byany method well-known to one of skill in the art, for example byimmunoassays. Antigenic epitopes need not necessarily be immunogenic.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder. A therapeutically effective amount may refer to the amountof therapeutic agent sufficient to delay or minimize the onset ofdisease, e.g., delay or minimize the spread of cancer. A therapeuticallyeffective amount may also refer to the amount of the therapeutic agentthat provides a therapeutic benefit in the treatment or management of adisease. Further, a therapeutically effective amount with respect to atherapeutic agent of the invention means the amount of therapeutic agentalone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of a disease.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of one or more symptoms ofa disorder in a subject as result of the administration of aprophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder. Afirst prophylactic or therapeutic agent can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks. 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent to asubject with a disorder.

“Effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toantibody dependent cell mediated cytotoxicity (ADCC), antibody dependentcell mediated phagocytosis (ADCP), and complement dependent cytotoxicity(CDC). Effector functions include both those that operate after thebinding of an antigen and those that operate independent of antigenbinding.

“Effector cell” as used herein is meant a cell of the immune system thatexpresses one or more Fc receptors and mediates one or more effectorfunctions. Effector cells include but are not limited to monocytes,macrophages, neutrophils, dendritic cells, eosinophils, mast cells,platelets, B cells, large granular lymphocytes, Langerhans' cells,natural killer (NK) cells, and may be from any organism including butnot limited to humans, mice, rats, rabbits, and monkeys.

“Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, staphylococcal protein A,streptococcal protein G, and viral FcγR. Fc ligands may includeundiscovered molecules that bind Fc.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 DECISION TREE FOR SELECTION OF Fc MUTANTS

An exemplary protocol for selecting Fc mutants.

FIG. 2 SCHEMATIC OF SEQUENCE OF 8B5.3.4 VARIABLE HEAVY CHAIN DOMAIN

Depiction of the of the 8B5.3.4 VH nucleotide and amino acid sequence(SEQ ID NOS:12 and 9, respectively).

FIG. 3 SCHEMATIC OF SEQUENCE OF 8B5.3.4 VARIABLE LIGHT CHAIN DOMAIN

Depiction of the 8B5.3.4 VL nucleotide and amino acid sequence (SEQ IDNOS:11 and 10, respectively).

FIG. 4 CAPTURE OF CH 4-4-20 ANTIBODY ON BSA-FITC SURFACE

6 μL of antibody at a concentration of approximately 20 μg/mL wasinjected at 5 μL/min over a BSA-fluoroscein isothiocyanate (FITC)surface. BIAcore sensogram of the binding of ch 4-4-20 antibodies withmutant Fc regions on the surface of the BSA-FITC immobilized sensor shipis shown. The marker was set on wild-type captured antibody response.

FIG. 5 SENSOGRAM OF REAL TIME BINDING OF FcγRIIIA TO CH 4-4-20ANTIBODIES CARRYING VARIANT Fc REGIONS

Binding of FcγRIIIA to ch-4-4-20 antibodies carrying variant Fc regionswas analyzed at 200 nM concentration. Responses were normalized at thelevel of ch-4-4-20 antibody obtained for wild-type.

Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L, A330S,K288N); Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11 (R255L, F243L);Mut 16 (F372Y); Mut 19 (K334N, K2461).

FIGS. 6 A-H ANALYSIS OF KINETIC PARAMETERS OF FcγRIIIA BINDING TOANTIBODIES CARRYING VARIANT Fc REGIONS

Kinetic parameters for FcγRIIIA binding to antibodies carrying variantFc regions were obtained by generating separate best fit curves for 200nM and 800 nM. Solid line indicates an association fit which wasobtained based on the k_(off) values calculated for the dissociationcurves in the 32-34 sec interval. K_(d) and k_(off) values represent theaverage from two concentrations.

FIG. 7 SENSOGRAM OF REAL TIME BINDING OF FcγRIIB-Fc FUSION PROTEINS TOANTIBODIES CARRYING VARIANT Fc REGIONS

Binding of FcγRIIB-Fc fusion proteins to ch-4-4-20 antibodies carryingvariant Fc regions was analyzed at 200 nM concentration. Responses werenormalized at the level of ch-4-4-20 antibody obtained for wild type.

FIGS. 8 A-C ANALYSIS OF KINETIC PARAMETERS FcγRIIB-Fc FUSION PROTEINS TOANTIBODIES CARRYING VARIANT Fc REGIONS

Kinetic parameters for FcγRIIB-Fc binding to antibodies carrying variantFc regions were obtained by generating separate best fit curves for 200nM and 800 nM. Solid line indicates an association fit which wasobtained based on the k_(off) values calculated for the dissociationcurves in the 32-34 sec. interval. K_(d) and K_(off) values representthe average from two concentrations.

Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L, A330S,K288N); Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11 (R255L, F243L);Mut 16 (F372Y); Mut 19 (K334N, K246I).

FIG. 9 RATIOS OF K_(off) (WT)/K_(off) (MUT) FOR FcγRIIIA-Fc PLOTTEDAGAINST ADCC DATA

Numbers higher than one show a decreased dissociation rate for FcγRIIIAbinding and increased dissociation rate for FcγRIIB-Fc binding relativeto wild-type. Mutants in the box have lower off rate for FcγRIIIAbinding and higher off rate for FcγRIIB-Fc binding.

FIG. 10 COMPETITION WITH UNLABELED FcγRIIIA

A kinetic screen was implemented to identify Fc region mutants withimproved K_(off) rates for binding FcγRIIIA. A library of Fc regionvariants containing P396L mutation was incubated with 0.1 μMbiotinylated FcγRIIIA-Linker-Avitag for one hour and then washed.Subsequently 0.8 uM unlabeled FcγRIIIA was incubated with the labeledyeast for different time points. Yeast was spun down and unlabeledFcγRIIIA was removed, Receptor bound yeast was stained with SA(streptavidin):PE (phycoerythrin) for FACS analysis.

FIGS. 11A-C FACS ANALYSIS BASED ON THE KINETIC SCREEN

Based on the calculated K_(off) from the data presented in FIG. 22, aone minute time point selection was chosen. A 10-fold excess of librarywas incubated with 0.1 μM biotinylated FcγRIIIA-Linker-Avitag monomer;cells were washed and incubated with unlabeled ligand for one minute;then washed and labeled with SA:PE. The cells were then sorted by FACS,selecting the top 0.3% binders. The nonselected P396L library wascompared to the yeast cells selected for improved binding by FACS. Thehistograms show the percentage of cells that are costained with bothFcγRIIIA/PE and goat anti-human Fc/FITC.

FIGS. 12 A-B SELECTION BASED ON SOLID PHASE DEPLETION OF FcγRIIB FcBINDERS

A. The P396L library was screened based on FcγRIIB depletion andFcγRIIIA selection using magnetic beads. The FcγRIIB depletion bymagnetic beads was repeated 5 times. The resulting yeast population wasanalyzed and found to show greater than 50% cell staining with goatanti-human Fc and a very small percentage of cells stained withFcγRIIIA. Subsequently cells were selected twice by FACS using 0.1biotinylated FcγRIIIA linker-avitag. Yeast cells were analyzed for bothFcγRIIIA and FcγRIIB binding after each sort and compared to wild typebinding.

B. Fc Mutants were selected from the FcγRIIB depleted yeast populationusing biotinylated FcγRIIIA 158F linker avitag monomer as a ligand. Thesort gate was set to select the top 0.25% FcγRIIIA 158F binders. Theresulting enriched population was analyzed by FACS for binding to thedifferent FcγRIIIA (158F and 158V), FcγRIIIB and FcγRIIA (131R).

FIG. 13 RELATIVE RATES OF SKBR3TARGET CELL LYSIS MEDIATED BY CHIMERIC4D5 HARBORING FC MUTANTS

Relative rates of lysis was calculated for each Fc mutant tested. Lysisrates for 4D5 antibody with Fc mutants were divided by the rate of lysismediated by wild type 4D5 antibody. Data from at least 2 independentassays were averaged and plotted on the histogram. For each Fc mutantdata from two different antibody concentrations are shown. The antibodyconcentrations were chosen to flank the point along the curve at whichlysis was ˜50%.

FIG. 14 RELATIVE RATES OF DAUDI CELL LYSIS MEDIATED BY CHIMERIC 2117HARBORING FC MUTANTS

Relative rates of lysis was calculated for each Fc mutant tested. Lysisrates for 2H7 antibody with Fc mutants were divided by the rate of lysismediated by wild type 2H7 antibody. Data from at least 1-2 independentassays were averaged and plotted on the histogram. For each Fc mutant,data from two different antibody concentrations are shown The antibodyconcentrations were chosen based on the point along the curve at whichlysis was ˜50%.

FIG. 15, Panels A-E Fc RECEPTOR PROFILES VIA FACS UPON CYTOKINETREATMENT OF MONOCYTES.

Cytokine treatment of monocytes increases low affinity Fc receptorexpression. Elutriated monocytes were cultured using specific cytokinesin serum free media. Fc receptor profiles were assayed using FACS.

FIG. 16 IMPROVED TUMOR CELL KILLING USING FC MUTANTS INMACROPHAGE-DERIVED MONOCYTES BASED ADCC.

Ch4D5 MAb concentration over 2 logs was tested using effector:targetratio of 35:1. Percent lysis was calculated as in FIG. 30.

FIG. 17 COMPLEMENT DEPENDENT CYTOTOXICITY ASSAY FLOW CHART.

The flow chart summarizes the CDC assays used.

FIG. 18 COMPLEMENT DEPENDENT CYTOTOXICITY ACTIVITY

Fc mutants that show enhanced binding to FcγRIIIA also showed improvedcomplement activity. Anti-CD20 ChMAb over 3 orders of magnitude wastitrated. Percent lysis was calculated as in as in FIG. 30.

FIG. 19 C1q BINDING TO 2B6 ANTIBODY

A. The diagram depicts the BIAcore format for analysis of 2B6 binding tothe first component of the complement cascade.

B. Sensogram of real time binding of 2B6 antibody carrying variant Fcregions to C1q.

FIGS. 20 A-D C1q BINDING TO 2B6 MUTANT ANTIBODY.

Sensogram of real time binding of 2B6 mutants to C1q (3.25 nM). Mutantsdepicted at MgFc51 (Q419H, P396L); MgFc51/60 in Panel A; MgFc55 andMgFc55/60 (Panel B), MgFc59 and MgFc59/60 (Panel C); and MgFc31/60(Panel D).

FIGS. 21A-D Fc VARIANTS WITH DECREASED BINDING TO FcγRIIB

Binding of FcR to ch4D5 antibodies to compare effect of D270E (60) onR255L, P396L double mutant (MgFc55). K_(D) was analyzed at differentconcentrations of FcR; 400 nM CD16A 158V; 800 nM CD16A 158F; 200 nMCD32B; 200 nM CD32A 131H. Analysis was performed using separate K_(D)using Biacore 3000 software.

FIGS. 22 A-D KINETIC CHARACTERISTICS OF 4D5 MUTANTS SELECTED FROMFcγRIIB DEPLETIONS/FcγRIIAH131 SELECTION

Binding of FcR to ch4D5 antibodies carrying different Fc mutationsselected by CD32B depletion and CD32A H131 screening strategy. K_(D) wasanalyzed at different concentrations of FcR; 400 nM CD16A 158V; 800 nMCD16A 158F; 200 nM CD32B; 200 nM CD32A 131H. Analysis was performedusing separate K_(D) using Biacore 3000 software.

FIG. 23. PLOT OF MDM ADCC DATA AGAINST THE K_(OFF) DETERMINED FOR CD32A131H BINDING AS DETERMINED BY BIACORE.

The mutants are as follows: MgFc 25 (E333A, K334A, S298A); MgFc68(D270E); MgFc38 (K392T, P396L); MgFc55 (R255L, P396L); MgFc31 (P247L,N421K); MgFc59(K370E, P396L).

FIGS. 24 A-B. ADCC ACTIVITY OF MUTANTS IN A HER2/NEU CHIMERIC MONOCLONALANTIBODY

Chimeric HER2/neu monoclonal antibodies containing mutant Fc regionswere assessed, in duplicate, for their ADCC activity and compared to theADCC activity of the wild type, chimeric Her2/neu antibody. The mutantsanalyzed are as follows: MGFc88 (F243L, R292P, Y300L, V305I, P396L),MGFc88A (F243L, R292P, Y300L, P396L), MGFc155 (F243L, R292P, Y300L).

FIGS. 25 A-B. ESTIMATED TUMOR WEIGHT IN MICE TREATED WITH WILD-TYPE ORFc MUTANT h2B6

Balb/c nude mice were inoculated subcutaneously with Daudi cells andadministered 25 μg, 2.5 μg or 0.25 μg weekly doses of either wild-typeh2B6 (A) or h2B6 harboring Fc mutant MGFc 0088 (F243L, R292P, Y300L,V305I, P396L) (B). Mice administered buffer alone were used as control.Tumor weight was calculated based on the estimated volume of thesubcutaneous tumor according to the formula (width²× length)/2.

FIGS. 26 A-B. SURVIVAL IN TUMOR BEARING MICE TREATED WITH WILD-TYPE ORFc MUTANT h2B6

Nude mice were inoculated with Daudi cells and administered 25 μg, 2.5μg or 0.25 μg weekly doses of either wild-type h2B6 (A) or h2B6harboring Fc mutant MGFc 0088 (F243L, R292P, Y300L, V305I, P396L) (B).Mice administered buffer alone were used as control.

FIG. 27. ESTIMATED TUMOR WEIGHT IN HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED h2B6 0088

mCD16−/− huCD16A+RAG1−/− C57BI/6 mice were inoculated subcutaneouslywith Raji cells and after two weeks were intraperitoneally administeredsix weekly doses of either buffer alone (PBS), or 250 μg, 25 μg or 2.5μg of wild-type h2B6 (Rituxan) or h2B6 comprising mutant FcMG0088(F243L. R292P, Y300L, V305I P396L) (MGA321). Tumor weight was calculatedbased on the estimated volume of the subcutaneous tumor according to theformula (width²× length)/2. Lines correspond to tumor weight, over time,for each individual mouse tested.

FIGS. 28 A-B. ESTIMATED TUMOR WEIGHT IN HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED h2B6 0088

mCD16−/− huCD16A+RAG1−/− C57BI/6 mice were inoculated subcutaneouslywith Raji cells and after three weeks were intraperitoneallyadministered five weekly doses of 250 μg, 25 μg or 2.5 μg of wild-typeh2B6 (Rituxan “Rituximab”) (A) or h2B6 comprising mutant FcMG0088(F243L, R292P, Y300L, V305I P396L) (h2B6 0088 “MGA321”) (B). Tumorweight was calculated based on the estimated volume of the subcutaneoustumor according to the formula (width² X length)/2.

FIG. 29. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED h2B6 31/60

mCD16−/− huCD16A+ nude (FoxN1) mice were intraperitoneally inoculatedwith EL4-CD32B cells and intraperitoneally administered on days 0, 1, 2,3 and 6 either wild-type h2B6 1.3 or h2B6 1.3 comprising mutant 31/60(P247L, D270E, N421K) (h2B6 1.3 3160). Mice administered buffer alone(PBS) were used as control.

FIG. 30. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED h2B6 31/60

mCD16−/− huCD16A+ nude (FoxN1) mice were intraperitoneally inoculatedwith EL4-CD32B cells and intraperitoneally administered on days 0-3 and6 doses of 10 μg/g body weight of either h2B6 comprising mutant 31/60(P247L, D270E, N421K) (h2B6 3160) or wild-type h2B6.

FIGS. 31A-B. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED h2B6 0088

(A) mCD16−/− huCD16A+ nude (FoxN1 or N/N) mice were intraperitoneallyinoculated with EL4-CD32B cells and intraperitoneally administered ondays 0-3 with doses of either h2B6 3.5 N297Q (negative control), h2B63.5 comprising mutant FcMG0088 (F243L, R292P, Y300L, V305I P396L) (h2B63.5 0088) or wild-type h2B6 3.5. Mice administered buffer alone (PBS)were used as control. (B) Tumor-bearing mice as in A wereintraperitoneally administered on days 0-3 and 6 doses of 4 μg/g bodyweight of either PBS, h2B6 comprising mutant FcMG0088 (F243L, R292P,Y300L, V305I P396L) (h2B6 0088) or wild-type h2B6.

FIG. 32. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED h2B6 0088

mCD16−/− huCD16A+hCD32A+ nude (FoxN1 or N/N) mice were intraperitoneallyinoculated with EL4-CD32B cells and intraperitoneally administered ondays 0-3 of either h2B6 3.5 N297Q (negative control), h2B6 comprisingmutant FcMG0088 (F243L, R292P, Y300L, V305I P396L) (h2B6 3.5 0088) orwild-type h2B6 3.5. Mice administered buffer alone (PBS) were used ascontrol.

FIGS. 33 A-C. SURVIVAL IN TUMOR-BEARING TRANSGENIC MICE TREATED WITHWILD-TYPE OR Fc-OPTIMIZED h2B6 0088

Nude (FoxN1) mice were intraperitoneally inoculated with EL4-CD32B cellsand intraperitoneally administered on days 0-3 of either h2B6 3.5 N297Q(negative control), h2B6 comprising mutant FcMG0088 (F243L, R292P,Y300L, V305I P396L) (h2B6 3.5 88) or wild-type h2B6 3.5. Miceadministered buffer alone (PBS) were used as control. The transgenicmice strains examined were (A) mCD16−/− huCD16A+, (B) mCD16−/− huCD16A+hCD32A+ and (C) mCD16−/− hCD32A+.

FIG. 34. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED AT VARYING INTERVALS WITH WILD-TYPE ORFc-OPTIMIZED h2B6 0088

mCD16−/− huCD16A+ nude (FoxN1 or N/N) mice were intraperitoneallyinoculated with EL4-CD32B cells and intraperitoneally treated with h2B6comprising mutant FcMG0088 (F243L, R292P, Y300L, V3051, P396L) (MGA321)at the indicated time intervals.

FIGS. 35 A-B. ESTIMATED TUMOR WEIGHT IN HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED ch4D5 0088

Nude (FoxN1) mice with the transgenic genotype mCD16−/− hCD16A+(A) ormCD16−/− hCD16A+hCD32A+(B) were inoculated subcutaneously with mSCOV3cells and, starting on day 0, were intraperitoneally administered eightweekly doses of either ch4D5 N297Q (negative control) or ch4D5comprising mutant FcMG0088 (F243L, R292P, Y300L, V305I, P396L) (ch4D50088). Mice administered buffer alone (PBS) were used as control. Tumorweight was calculated based on the estimated volume of the subcutaneoustumor according to the formula (width²× length)/2.

FIGS. 36 A-B. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED ch4D5 0088

mCD16−/− huCD16A+ nude (N/N) mice were intraperitoneally inoculated withmSKOV3 cells and, starting on day 0, were intraperitoneally administeredsix weekly doses of either 100 μg (A) or 1 μg (B) of either wild-typech4D5, ch4D5 N297Q (negative control) or ch4D5 comprising mutantFcMG0088 (F243L, R292P, Y300L, V305I P396L) (ch4D5 0088). Miceadministered buffer alone (PBS) were used as control.

FIGS. 37 A-B. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED VARIANTS OF ch4D5

mCD16−/− huCD16A+ nude (N/N) mice were intraperitoneally inoculated withmSKOV3 cells and, starting on day 0, were intraperitoneally administeredeight weekly doses of either 100 μg (A) or 10 μg (B) of either wild-typech4D5, ch4D5 N297Q (negative control), ch4D5 comprising mutant FcMG0088(F243L, R292P, Y300L, V305I P396L) (ch4D5 0088), ch4D5 mutant MGFc0155(F243L, R292P, Y300L) (ch4D5 0155) or ch4D5 mutant MCFc3160 (P247L,D270E, N421K) (“ch4D5 3160”). Mice administered buffer alone (PBS) wereused as control.

FIGS. 38 A-B. SURVIVAL IN TUMOR-BEARING HUMAN Fc RECEPTOR-EXPRESSINGTRANSGENIC MICE TREATED WITH WILD-TYPE OR Fc-OPTIMIZED ch4D5 0088

Nude (N/N) mice with the transgenic genotype mCD16−/− hCD16A+(A) ormCD16−/− hCD16A+hCD32A+(B) were intraperitoneally inoculated with mSKOV3cells and, starting on day 0, intraperitoneally administered eightweekly doses of either ch4D5 comprising mutant FcMG0088 (F243L, R292P,Y300L, V305I P396L) (ch4D5 0088) or ch4D5 N297Q (negative control). Miceadministered buffer alone (PBS) were used as control.

FIG. 39 A-D. Fc OPTIMIZATION ENHANCES TUMOR-CELL DEPLETION IN VIVO.

(A-B) Enhanced reduction of tumor burden by Fc-engineered hu2B6treatment of Daudi cell subcutaneous xenografts in Balb/c FoxN1 (nu/nu)mice (6-8 mice/group). Statistical significance between curves wasdetermined by a student T test; WT vs MG12, P=0.431; WT vs MG4, P=0.002;MG4 vs MG12, P=0.002. (C-D) Kaplan-Meier survival plots of mFcγRIII−/−human CD16A+ FoxN1 mice injected intra-peritoneally with CD32B-EL4 cells(10 mice/group) and treated with either WT-Fc hu2B6 or the indicatedFc-engineered forms of hu2B6 (C, 10 μg/g; D, 4 μg/g). Data was analyzedfor significance using log-rank analysis.

FIG. 40. GLYCOSYLATION PATTERNS OF ANTIBODIES HAVING ALTERED FC REGIONS.

(Panels A-D) show the results of investigations into the glycosylationpatterns of the antibodies of the present invention (plotted is detectedsignal in lumens vs. chromatographic elution time in minutes). In allpanels, ▪ is GlcNAc; ● is galactose; ◯ is mannose and Δ is fucose. PanelA shows the assignment of N-linked oligosaccharides as determined usingan antibody reference panel. Panels B and C show the results on twopreparations of antibody ch45D4-FcMT2. Panel D is a digest control.Arrows indicate the assignments of glycosylation patterns to peaks;dashed arrows indicate the expected positions of the glycosylationpatterns of Panel A.

FIG. 41. GlcNAc₂Man₉ (“Man9”) GLYCOSYLATION STRUCTURE.

The GlcNAc₂Man₉ (“Man9”) glycosylation structure. Cleavage of mannoseunits results in the formation of GlcNAc₂Man₈ (“Man8”), GlcNAc₂Man₇(“Man7”), GlcNAc₂Man₆ (“Man6”) and GlcNAc₂Man₅ (“Man5”) structures.

FIG. 42. GlcNAc₂Man₅ (“Man5”) GLYCOSYLATION STRUCTURE.

The GlcNAc₂Man₅ (“Man5”) structure is processed by the successive actionof glycosyltransferases to generate “G0F,” “G1F” and “G2F”oligosaccharides.

FIG. 43. GLYCOSYLATION STRUCTURE AND FcγR BINDING OF FC TETRA VARIANT:F243X, R292P, Y300L, P396L

The Figure shows the effect of altering the identity of the residuesubstituted at position F243 of a tetra variant (F243X, R292P, Y300L,P396L) on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors, CD16A, CD32A and CD32B.

FIG. 44. GLYCOSYLATION STRUCTURE AND FcγR BINDING OF FC TETRA VARIANT:F243L, R292X, Y300L, P396L

The Figure shows the effect of altering the identity of the residuesubstituted at position 8292 of a tetra variant (F243L, R292X, Y300L,P396L) on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors, CD16A, CD32A and CD32B.

FIG. 45. EFFECT OF PROGRESSIVE ALTERATIONS OF RESIDUES R292, Y300 ANDP396 OF AN F243C Fc VARIANT

The Figure shows the effect of progressively altering the identity ofthe residue substituted at position R292, Y300 and P396 of an F243C Fcvariant on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors, CD16A, CD32A and CD32B.

FIG. 46. EFFECT OF PROGRESSIVE ALTERATIONS OF RESIDUES R292, Y300 ANDP396 OF AN F243C Fc VARIANT

The Figure shows the effect of progressively altering the identity ofthe residue substituted at position R292, Y300 and P396 of an F243L Fcvariant on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors, CD16A, CD32A and CD32B.

FIG. 47. EFFECT OF PROGRESSIVE ALTERATIONS OF RESIDUES R292, Y300 ANDP396 OF AN F243R Fc VARIANT

The Figure shows the effect of progressively altering the identity ofthe residue substituted at position R292, Y300 and P396 of an F243R Fcvariant on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors, CD16A, CD32A and CD32B.

FIG. 48. EFFECT OF PROGRESSIVE ALTERATIONS OF RESIDUES R292, Y300 ANDP396 OF AN F243V Fc VARIANT

The Figure shows the effect of progressively altering the identity ofthe residue substituted at position R292, Y300 and P396 of an F243V Fcvariant on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors, CD16A, CD32A and CD32B.

6. DETAILED DESCRIPTION

The present invention relates to methods of treatment of cancer or otherdiseases using molecules, preferably polypeptides, and more preferablyimmunoglobulins (e.g., antibodies), comprising a variant Fc region,having one or more amino acid modifications (e.g., substitutions, butalso including insertions or deletions) in one or more regions, whichmodifications alter, e.g., increase or decrease, the affinity of thevariant Fc region for an FcγR. Enhancing the ability of immunoglobulinsto mediate antibody-dependent cell-mediated cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated phagocytosis (ADCP) provides an approach for enhancing thetherapeutic activity of immunoglobulins against cancers and infectiousdiseases.

The invention thus encompasses therapeutic antibodies in the treatmentor prevention of a disease or disorder, or the amelioration of a symptomthereof, where an enhanced efficacy of effector cell function (e.g.,ADCC) mediated by FcγR is desired, e.g., cancer or infectious disease,or where a modulation of effector cell function mediated by FcγR isdesired, e.g., autoimmune or inflammatory disorders. In someembodiments, the invention encompasses the use of molecules comprisingFc regions with amino acid modifications including but not limited toany of the modifications disclosed in U.S Patent ApplicationPublications 2005/0037000 and 2005/0064514; and International PatentApplication Publication WO 04/063351. Each of the above mentionedapplications is incorporated herein by reference in its entirety.

The polypeptides of the present invention may have variant Fc domains.Modification of the Fc domain normally leads to an altered phenotype,for example altered serum half-life, altered stability, alteredsusceptibility to cellular enzymes or altered effector function. It maybe desirable to modify the antibody of the invention with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. Reduction or elimination of effectorfunction is desirable in certain cases, for example in the case ofantibodies whose mechanism of action involves blocking or antagonism,but not killing of the cells bearing a target antigen. Increasedeffector function is generally desirable when directed to undesirablecells, such as tumor and foreign cells, where the FcγRs are expressed atlow levels, for example, tumor specific B cells with low levels ofFcγRIIB (e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma). Insaid embodiments, molecules of the invention with conferred or alteredeffector function activity are useful for the treatment and/orprevention of a disease, disorder or infection where an enhancedefficacy of effector function activity is desired.

In certain embodiments, the molecules of the invention comprise one ormore modifications to the amino acids of the Fc domain, which reduce theaffinity and avidity of the Fc region and, thus, the molecule of theinvention, for one or more FcγR receptors. In other embodiments, themolecules of the invention comprise one or more modifications to theamino acids of the Fc region, which increase the affinity and avidity ofthe Fc region and, thus, the molecule of the invention, for one or moreFcγR receptors. In other embodiments, the molecules comprise a variantFc domain wherein said variant confers or mediates increased ADCCactivity and/or an increased binding to FcγRIIA, relative to a moleculecomprising no Fc domain or comprising a wild-type Fc domain. Inalternate embodiments, the molecules comprise a variant Fc domainwherein said variant confers or mediates decreased ADCC activity (orother effector function) and/or an increased binding to FcγRIIB,relative to a molecule comprising no Fc domain or comprising a wild-typeFc domain.

In some embodiments, the invention encompasses molecules comprising avariant Fc region, which variant Fc region does not show a detectablebinding to any FcγR, relative to a comparable molecule comprising thewild-type Fc region. In other embodiments, the invention encompassesmolecules comprising a variant Fc region, which variant Fc region onlybinds a single FcγR, preferably one of FcγRIIA, FcγRIIB, or FcγRIIIA.

The polypeptides of the present invention may comprise alteredaffinities for an activating and/or inhibitory Fcγ receptor. In oneembodiment, the antibody or polypeptide comprises a variant Fc regionthat has increased affinity for FcγRIIB and decreased affinity forFcγRIIIA and/or FcγRIIA, relative to a comparable molecule with awild-type Fc region. In another embodiment, the polypeptides of thepresent invention comprise a variant Fc region, which has decreasedaffinity for FcγRIIB and increased affinity for FcγRIIIA and/or FcγRIIA,relative to a comparable molecule with a wild-type Fc region. In yetanother embodiment, the polypeptides of the present invention comprise avariant Fc region that has decreased affinity for FcγRIIB and decreasedaffinity for FcγRIIIA and/or FcγRIIA, relative to a comparable moleculewith a wild-type Fc region. In still another embodiment, thepolypeptides of the present invention comprise a variant Fc region,which has unchanged affinity for FcγRIIB and decreased (or increased)affinity for FcγRIIIA and/or FcγRIIA, relative to a comparable moleculewith a wild-type Fc region.

In certain embodiments, the invention encompasses immunoglobulinscomprising a variant Fc region with an altered affinity for FcγRIIIAand/or FcγRIIA such that the immunoglobulin has an enhanced effectorfunction, e.g., antibody dependent cell mediated cytotoxicity.Non-limiting examples of effector cell functions includeantibody-dependent cell mediated cytotoxicity (ADCC), antibody-dependentphagocytosis, phagocytosis, opsonization, opsonophagocytosis, cellbinding, rosetting, C1q binding, and complement dependent cell mediatedcytotoxicity.

In a preferred embodiment, the alteration in affinity or effectorfunction is at least 2-fold, preferably at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least9-fold, at least 10-fold, at least 50-fold, or at least 100-fold,relative to a comparable molecule comprising a wild-type Fc region. Inother embodiments of the invention, the variant Fc regionimmunospecifically binds one or more FcRs with at least 65%, preferablyat least 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%,225%, or 250% greater affinity relative to a molecule comprising awild-type Fc region. Such measurements can be in vivo or in vitroassays, and in a preferred embodiment are in vitro assays such as ELISAor surface plasmon resonance assays.

In different embodiments, the molecules comprise a variant Fc domainwherein said variant agonizes at least one activity of an FcγR receptor,or antagonizes at least one activity of an FcγR receptor. In a preferredembodiment, the molecules comprise a variant that agonizes (orantagonizes) one or more activities of FcγRIIB, for example, B cellreceptor-mediated signaling, activation of B cells, B cellproliferation, antibody production, intracellular calcium influx of Bcells, cell cycle progression, FcγRIIB-mediated inhibition of FcεRIsignaling, phosphorylation of FcγRIIB, SHIP recruitment, SHIPphosphorylation and association with Shc, or activity of one or moredownstream molecules (e.g., MAP kinase, JNK, p38, or Akt) in the FcγRIIBsignal transduction pathway. In another embodiment, the moleculescomprise a variant that agonizes (or antagonizes) one or more activitiesof FcεRI, for example, mast cell activation, calcium mobilization,degranulation, cytokine production, or serotonin release.

In certain embodiments, the molecules comprise an Fc domain comprisingdomains or regions from two or more IgG isotypes (e.g., IgG1, IgG2, IgG3and IgG4). The various IgG isotypes exhibit differing physical andfunctional properties including serum half-life, complement fixation,FcγR binding affinities and effector function activities (e.g. ADCC,CDC, etc.) due to differences in the amino acid sequences of their hingeand/or Fc domains, for example as described in Flesch and Neppert (1999)J. Clin. Lab. Anal. 14:141-156; Chappel et al. (1993) J. Biol. Chem.33:25124-25131; Chappel et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.)88:9036-9040; Bruggemann et al. (1987) J. Exp. Med. 166:1351-1361. Thistype of variant Fc domain may be used alone, or in combination with anamino acid modification, to affect Fc-mediated effector function and/orbinding activity. In combination, the amino acid modification and IgGhinge/Fc region may display similar functionality (e.g., increasedaffinity for FcγRIIA) and may act additively or, more preferably,synergistically to modify the effector functionality in the molecule ofthe invention, relative to a molecule of the invention comprising awild-type Fc region. In other embodiments, the amino acid modificationand IgG Fc region may display opposite functionality (e.g., increasedand decreased affinity for FcγRIIA, respectively) and may act toselectively temper or reduce a specific functionality in the molecule ofthe invention, relative to a molecule of the invention not comprising anFc region or comprising a wild-type Fc region of the same isotype.

In specific embodiments, the invention encompasses therapeuticantibodies that immunospecifically bind FcγRIIB via their variabledomains with greater affinity than FcγRIIA, e.g., antibodies derivedfrom mouse monoclonal antibody produced by clone 2B6 or 3H7, having ATCCaccession numbers PTA-4591 and PTA-4592, respectively. In otherembodiments, the anti-FcγRIIB antibodies of the invention are derivedfrom a mouse monoclonal antibody produced by clone 1D5, 2E1, 2H9, 2D11,or 1F2, having ATCC Accession numbers, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively. Hybridomas producing antibodies2B6 and 3H7 have been deposited with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on Aug.13, 2002 under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedures, and assigned accession numbers PTA-4591(for hybridoma producing 2B6) and PTA-4592 (for hybridoma producing3H7), respectively, and are incorporated herein by reference. Hybridomasproducing 1 D5, 2E1, 2H9, 2D11, and 1F2 were deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on May 7,2004, and assigned accession numbers PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively and are incorporated herein byreference. In preferred embodiments, the anti-FcγRIIB antibodies of theinvention (e.g., 2B6) comprise a variant Fc region, wherein said variantFc region has a substitution at portion at position 243 with leucine, atposition 292 with proline, at position 300 with leucine, at position 305with isoleucine, and at position 396 with leucine. In anotherembodiment, the invention encompasses a 2B6 antibody having a leucine atposition 243, a proline at position 292, a leucine at position 300, anisoleucine at position 305, and a leucine at position 396. In otherembodiments, the invention encompasses a 2B6 antibody comprising avariant Fc region having a substitution at position 243 with leucine, atposition 292 with proline, and at position 300 with leucine. In otherembodiments, the invention encompasses a 2B6 antibody having a leucineat position 243, a proline at position 292, and a leucine at position300. In other embodiments, the invention encompasses a 2B6 antibodycomprising a variant Fc region having a substitution at position 247with leucine, at position 270 with glutamic acid, and at position 421with lysine. In another embodiment, the invention encompasses a 2B6antibody having a leucine at position 247, a glutamic acid at position292, and a lysine at position 421.

In a specific embodiment, the invention encompasses therapeuticantibodies that bind the Her2/neu protooncogene (amino acid sequence SEQID NO:31) (e.g., Ab4D5 antibody as disclosed in Carter et al., 1992,Proc. Natl. Acad. Sci. USA 89:4285-9; U.S. Pat. No. 5,677,171; orInternational Patent Application Publication WO 01/00245, each of whichis hereby incorporated by references in its entirety). In a certainembodiment, the 4D5 antibody is chimeric. In another embodiment, the 4D5antibody is humanized. In a specific embodiment, the 4D5 antibody isengineered to comprise a variant Fc region by modification (e.g.,substitution, insertion, deletion) of at least one amino acid residuewhich modification increases the affinity of the Fc region for FcγRIIIAand/or FcγRIIA and/or decreases the affinity of the Fc region forFcγRIIB and/or modulates the effector function of the antibody relativeto a comparable antibody comprising a wild-type Fc region. In certainembodiments, the 4D5 antibody of the invention comprises a variant Fcregion having a substitution at position 243 with leucine, at position292 with proline, at position 300 with leucine, at position 305 withisoleucine, and at position 396 with leucine. In other embodiments, theinvention encompasses a 4D5 antibody having a leucine at position 243, aproline at position 292, a leucine at position 300, an isoleucine atposition 305, and a leucine at position 396. In yet another embodiment,the invention encompasses a 4D5 antibody comprising a variant Fc regionhaving a substitution at position 243 with leucine, at position 292 withproline, and at position 300 with leucine. In still other embodiments,the invention encompasses a 4D5 antibody having a leucine at position243, a proline at position 292, and a leucine at position 300. In otherembodiments, the invention encompasses a 4D5 antibody comprising avariant Fc region having a substitution at position 247 with leucine, atposition 270 with glutamic acid, and at position 421 with lysine. Inanother embodiment, the invention encompasses a 4D5 antibody having aleucine at position 247, a glutamic acid at position 292, and a lysineat position 421.

In some embodiments, the invention encompasses molecules comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region does not show a detectable binding to any FcγR (e.g., does notbind FcγRIIA, FcγRIIB, or FcγRIIIA, as determined by, for example, anELISA assay), relative to a comparable molecule comprising the wild-typeFc region.

In a specific embodiment, the invention encompasses molecules comprisinga variant Fc region, wherein said variant Fc region comprises at leastone amino acid modification relative to a wild type Fc region, whichvariant Fc region only binds one FcγR, wherein said FcγR is FcγIIIA. Inanother specific embodiment, the invention encompasses moleculescomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild type Fc region,which variant Fc region only binds one FcγR, wherein said FcγR isFcγRIIA. In yet another embodiment, the invention encompasses moleculescomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild type Fc region,which variant Fc region only binds one FcγR, wherein said FcγR isFcγRIIB. The invention particularly relates to the modification of humanor humanized therapeutic antibodies (e.g., tumor specificanti-angiogenic or anti-inflammatory monoclonal antibodies) forenhancing the efficacy of therapeutic antibodies by enhancing, forexample, the effector function of the therapeutic antibodies, e.g.,enhancing ADCC.

In certain embodiments, the molecules comprise a variant Fc region,having one or more amino acid modifications in one or more regions,which modification(s) alter (relative to a wild-type Fc region) theRatio of Affinities of the variant Fc region to an activating FcγR (suchas FcγRIIA or FcγRIIIA) relative to an inhibiting FcγR (such asFcγRIIB):

${{Ratio}\mspace{14mu}{of}\mspace{14mu}{Affinities}} = \frac{{Wild}\text{-}{Type}{\mspace{11mu}\;}{to}\mspace{14mu}{Variant}\mspace{14mu}{Change}\mspace{14mu}{in}\mspace{14mu}{Affinity}\mspace{14mu}{to}\mspace{14mu}{Fc}\;\gamma\; R_{Activating}}{{Wild}\text{-}{Type}\mspace{14mu}{to}\mspace{14mu}{Variant}\mspace{14mu}{Change}\mspace{14mu}{in}\mspace{14mu}{Affinity}\mspace{14mu}{to}\mspace{14mu}{Fc}\;\gamma\; R_{Inhibiting}}$

Where an Fc variant has a Ratio of Affinities greater than 1, themethods of the invention have particular use in providing a therapeuticor prophylactic treatment of a disease, disorder, or infection, or theamelioration of a symptom thereof, where an enhanced efficacy ofeffector cell function (e.g., ADCC) mediated by FcγR is desired, e.g.,cancer or infectious disease. Where an Fc variant has a Ratio ofAffinities less than 1, the methods of the invention have particular usein providing a therapeutic or prophylactic treatment of a disease ordisorder, or the amelioration of a symptom thereof, where a decreasedefficacy of effector cell function mediated by FcγR is desired, e.g.,autoimmune or inflammatory disorders. Table 1 lists exemplary single,double, triple, quadruple and quintuple mutations by whether their Ratioof Affinities is greater than or less than 1. Specific binding data forvarious mutations is listed in Table 2, and more information concerningthese mutations may be found in the Antibody Engineering Technology Art.

TABLE 1 Exemplary Single and Multiple Mutations Listed by Ratio ofAffinities Ratio Single Double Triple Quadruple Quintuple >1 F243L F243L& F243L, P247L & L234F, F243L, R292P & L235V, F243L, D270E R292P N421KY300L R292P, Y300L R292G F243L & F243L, R292P & L235I, F243L, R292P & &P396L R292P Y300L Y300L Y300L L235P, F243L, F243L & F243L, R292P &L235Q, F243L, R292P & R292P, Y300L P396L V305I Y300L & P396L D270E &F243L, R292P & F243L, P247L, D270E & F243L, R292P, P396L P396L N421KV305I, Y300L R292P & F243L, Y300L & F243L, R255L, D270E & & P396L Y300LP396L P396L R292P & P247L, D270E & F243L, D270E, G316D V305I N421K &R416G R292P & R255L, D270E & F243L, D270E, K392T P396L P396L & P396LY300L & D270E, G316D & F243L, D270E, P396L & P396L R416G Q419H P396L &D270E, K392T & F243L, R292P, Y300L, Q419H P396L & P396L D270E, P396L &F243L, R292P, V305I & Q419H P396L V284M, R292L & P247L, D270E, Y300LK370N & N421K R292P, Y300L & R255L, D270E, R292G P396L & P396L R255L,D270E, Y300L & P396L D270E, G316D, P396L & R416G <1 Y300L F243L & F243L,R292P & P396L P396L V305I P247L & N421K R255L & P396L R292P & V305IK392T & P396L P396L & Q419H

TABLE 2 Detailed Binding Information for Exemplary Fc Variants Ratio ofAffinities CD16A/ CD16A CD16A CD32B Fc sequence V158 F158 CD32B V158F158 Ratio of Affinities >1 Class I: Increased Binding to CD16;Decreased Binding to CD32B F243L 4.79 3.44 0.84 5.70 4.10 F243L P247LD270E 2.30 3.45 0.32 7.19 10.78 N421K F243L P247L N421K 1.89 1.71 0.1711.12 10.06 F243L R255L D270E 1.75 1.64 0.38 4.61 4.32 P396L F243L D270EG316D 1.50 1.34 0.20 7.50 6.70 R416G F243L D270E K392T 3.16 2.44 0.447.18 5.55 P396L F243L D270E P396L 1.46 1.15 0.26 5.62 4.42 Q419H F243LR292P 4.73 0.12 39.4 F243L R292P 4 1.67 0.16 25 10.44 F243L R292P P300L6.69 2.3 0.32 20.9 7.19 F243L R292P V305I 2.56 1.43 ND >25 >25 F243LR292P V305I 5.37 2.53 0.40 13.43 6.33 P396L P247L D270E N421K 1.89 2.460.58 3.26 4.24 R255L D270E R292G 1.39 1.30 0.65 2.14 2.00 P396L R255LD270E Y300L 1.52 1.74 0.87 1.75 2.00 P396L R255L D270E P396L 1.34 1.650.87 1.54 1.90 D270E 1.25 1.48 0.39 3.21 3.79 D270E G316D R416G 2.182.49 0.78 2.79 3.19 D270E K392T P396L 1.81 2.28 0.79 2.29 2.89 D270EP396L 1.38 1.65 0.89 1.55 1.85 D270E P396L G316D 1.22 1.07 1.14 R416GD270E P396L Q419H 1.64 2.00 0.68 2.41 2.94 V284M R292P K370N 1.14 1.370.37 3.1 3.7 R292G 1.54 0.25 6.2 R292P 2.90 0.25 11.60 R292P V305I 1.321.28 0.37 3.6 3.46 Class II: Decreased Binding to CD16; GreatlyDecreased Binding to CD32B R292P 0.64 0.25 2.56 R292P F243L 0.6 0.125.00 Class III: Increased Binding to CD16; Unchanged Binding to CD32BF243I R292P Y300L 10.9 3.12 1.05 10.4 2.97 V305I P396L F243L R292P Y300L10.06 5.62 1.07 9.40 5.25 P396L R292P V305I P396L 1.85 1.90 0.92 2.012.07 Class IV: Greatly Increased Binding to CD16; Increased Binding toCD32B F243L R292P Y300L 10.06 8.25 1.38 7.29 5.98 V305I P396L D270EG316D P396L 1.22 1.07 1.14 R416G Ratio of Affinities <1 Class V:Unchanged Binding to CD16; Increased Binding to CD32B R255L P396L 1.092.22 0.49 Y300L 1.01 1.18 0.99 Class VI: Increased Binding to CD16;Greatly Increased Binding to CD32B F243L P396L 1.49 1.60 2.22 0.67 0.72P247L N421K 1.29 1.73 2.00 0.65 0.87 R255L P396L 1.39 2.22 0.49 0.63R292P V305I 1.59 2.11 2.67 0.60 0.79 K392T P396L 1.49 1.81 2.35 0.630.77 P396L 1.27 1.73 2.58 0.49 0.67 P396L Q419H 1.19 1.19 1.33 0.89 0.89Class VII: Decreased Binding to CD16; Increased/Unchanged Binding toCD32B D270E G316D P396L 0.94 1.07 0.88 R416G

In other embodiments, the molecules comprise a variant Fc region havingone or more amino acid substitutions, which substitutions alter(relative to a wild-type Fc region) the binding of the variant Fcregion, e.g., enhance the binding to an activating FcγR (such as FcγRIIAor FcγRIIIA) and/or reduce the binding to an inhibiting FcγR (such asFcγRIIB). Various Fc mutations having one or more amino acid changeswere engineered and analyzed by surface plasmon resonance for k_(off),as shown in Table 3. Dissociation rate constants for binding the variousFcγR were determined by BIAcore analysis and directly compared withthose for the wild-type Fc, with the ratio (x=WT k_(off)/mutant k_(off))indicated in the right-hand columns of Table 3 with respect to each FcγRtested.

TABLE 3 Comparison Of k_(off) Of Fc Mutants to Wild-Type Fc

Abbreviations: M, Mutant Number; nd, no detectable binding; nt, nottested. Values with ≧80% difference (≧0.8 fold) from wild-type in eitherdirection are in bold. Shading denotes Fc mutants identified directly byyeast display; all other mutants were constructed by site-directedmutagenesis.

There is also extensive guidance in the Antibody Engineering TechnologyArt concerning desirable modifications. Exemplary modifications that maybe desirable in certain circumstances are listed below:

In a specific embodiment, in variant Fc regions, any amino acidmodifications (e.g., substitutions) at any of positions 235, 240, 241,243, 244, 247, 262, 263, 269, 298, 328, or 330 and preferably one ormore of the following residues: A240, I240, L241, L243, H244, N298, I328or V330. In a different specific embodiment, in variant Fc regions, anyamino acid modifications (e.g., substitutions) at any of positions 268,269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305,307, 309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419,430, 434, 435, 437, 438 or 439 and preferably one or more of thefollowing residues: H280, Q280, Y280, G290, S290, T290, Y290, N294,K295, P296, D298, N298, P298, V298, I300 or L300.

In a preferred embodiment, in variant Fc regions that bind an FcγR withan altered affinity, any amino acid modifications (e.g., substitutions)at any of positions 255, 256, 258, 267, 268, 269, 270, 272, 276, 278,280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301,303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334,335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435,437, 438 or 439. Preferably, the variant Fc region has any of thefollowing residues: A256, N268, Q272, D286, Q286, S286, A290, 5290,A298, M301, A312, E320, M320, Q320, R320, E322, A326, D326, E326, N326,S326, K330, T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335,Q335, A359, A360 or A430.

In a different embodiment, in variant Fc regions that bind an FcγR (viaits Fc region) with a reduced affinity, any amino acid modifications(e.g., substitutions) at any of positions 252, 254, 265, 268, 269, 270,278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327,329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434,435, 437, 438 or 439.

In a different embodiment, in variant Fc regions that bind an FcγR (viaits Fc region) with an enhanced affinity, any amino acid modifications(e.g., substitutions) at any of positions 280, 283, 285, 286, 290, 294,295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340,360, 378, 398 or 430. In a different embodiment, in variant Fc regionsthat binds FcγRIIA with an enhanced affinity, any of the followingresidues: A255, A256, A258, A267, A268, N268, A272, Q272, A276, A280,A283, A285, A286, D286, Q286, 5286, A290, S290, M301, E320, M320, Q320,R320, E322, A326, D326, E326, S326, K330, A331, Q335, A337 or A430.

In other embodiments, the invention encompasses the use of any Fcvariant known in the art, such as those disclosed in Jefferis et al.(2002) Immunol Lett 82:57-65; Presta et al. (2002) Biochem Soc Trans30:487-90; Idusogie et al. (2001) J Immunol 166:2571-75; Shields et al.(2001) J Biol Chem 276:6591-6604; Idusogie et al. (2000) J Immunol164:4178-84; Reddy et al. (2000) J Immunol 164:1925-33; Xu et al. (2000)Cell Immunol 200:16-26; Armour et al. (1999) Eur J Immunol 29:2613-24;Jefferis et al. (1996) Immunol Lett 54:101-04; Hinton et al; 2004, J.Biol. Chem. 279(8): 6213-6; Lund et al. (1996) J Immunol 157:4963-69;Hutchins et al. (1995) Proc. Natl. Acad. Sci. (U.S.A.) 92:11980-84;Jefferis et al. (1995) Immunol Lett. 44:111-17; Lund et al. (1995) FASEBJ 9:115-19; Alegre et al. (1994) Transplantation 57:1537-43; Lund et al.(1992) Mol Immunol 29:53-59; Lund et al. (1991) J. Immunol. 147:2657-62;Duncan et al. (1988) Nature 332:563-64; U.S. Pat. Nos. 5,624,821;5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publications WO00/42072 and PCT WO 99/58572.

Preferred variants include one or more modifications at any ofpositions: 228, 230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244,245, 247, 262, 263, 264, 265, 266, 271, 273, 275, 281, 284, 291, 296,297, 298, 299, 302, 304, 305, 313, 323, 325, 326, 328, 330 or 332.

Particularly preferred variants include one or more modificationsselected from groups A-AI:

-   -   A. 228E, 228K, 228Y or 228G;    -   B. 230A, 230E, 230Y or 230G;    -   C. 231E, 231K, 231Y, 231P or 231G;    -   D. 232E, 232K, 232Y, 232G;    -   E. 233D;    -   F. 234I or 234F;    -   G. 235D, 235Q, 235P, 235I or 235V;    -   H. 239D, 239E, 239N or 239Q;

I. 240A, 240I, 240M or 240T;

-   -   J. 243R, 243, 243Y, 243L, 243Q, 243W, 243H or 243I;    -   K. 244H;    -   L. 245A;    -   M. 247G, 247V or 247L;    -   N. 262A, 262E, 2621, 262T, 262E or 262F;    -   O. 263A, 263I, 263M or 263T;    -   P. 264F, 264E, 264R, 264I, 264A, 264T or 264W;    -   Q. 265F, 265Y, 265H, 265I, 265L, 265T, 265V, 265N or 265Q;    -   R. 266A, 266I, 266M or 266T;    -   S. 271D, 271E, 271N, 271Q, 271K, 271R, 271S, 271T, 271H, 271A,        271V, 271L, 271I, 271F, 271M, 271Y, 271W or 271G;    -   T. 2731;    -   U. 275L or 275W;

V. 281D, 281K, 281Y or 281P;

-   -   W. 284E, 284N, 284T, 284L, 284Y or 284M;

X. 291D, 291E, 291Q, 291T, 291H, 2911 or 291G;

-   -   Y. 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M,        299N, 299P, 299Q, 299R, 299S, 299V, 299W or 299Y;    -   Z. 302I;    -   AA. 304D, 304N, 304T, 304H or 304L    -   AB. 305I;    -   AC. 313F;    -   AD. 323I;    -   AE. 325A, 325D, 325E, 325G, 325H, 325I, 325L, 325K, 325R, 325S,        325F, 325M, 325T, 325V, 325Y, 325W or 325P;    -   AF. 328D, 328Q, 328K, 328R, 328S, 328T, 328V, 328I, 328Y, 328W,        328P, 328G, 328A, 328E, 328F, 328H, 328M or 328N;    -   AG. 330L, 330Y, 330I or 330V;    -   AH. 332A, 332D, 332E, 332H, 332N, 332Q, 332T, 332K, 332R, 332S,        332V, 332L, 332F, 332M, 332W, 332P, 332G or 332Y; and    -   AI. 336E, 336K or 336Y.

Still more particularly preferred variants include one or moremodifications selected from Groups 1-105 of Table 4:

TABLE 4 Group Variant 1 A330L/I332E 2 D265F/N297E/I332E 3D265Y/N297D/I332E 4 D265Y/N297D/T299L/I332E 5 F241E/F243Q/V262T/V264F 6F241E/F243Q/V262T/V264E/ I332E 7 F241E/F243R/V262E/V264R 8F241E/F243R/V262E/V264R/ I332E 9 F241E/F243Y/V262T/V264R 10F241E/F243Y/V262T/V264R/ I332E 11 F241L/F243L/V262I/V264I 12 F241L/V262I13 F241R/F243Q/V262T/V264R 14 F241R/F243Q/V262T/V264R/ I332E 15F241W/F243W/V262A/V264A 16 F241Y/F243Y/V262T/V264T 17F241Y/F243Y/V262T/V264T/ N297D/I332E 18 F243L/V262I/V264W 19 P243L/V264I20 L328D/I332E 21 L328E/I332E 22 L328H/I332E 23 L328I/I332E 24L328M/I332E 25 L328N/I332E 26 L328Q/I332E 27 L328T/I332E 28 L328V/I332E29 N297D/A330Y/I332E 30 N297D/I332E 31 N297D/I332E/S239D/A330L 32N297D/S298A/A330Y/I332E 33 N297D/T299L/I332E 34 N297D/T299F/I332E/N297D/T299H/I332E 35 N297D/T299I/I332E 36 N297D/T299L/I332E 37N297D/T299V/I332E 38 N297E/I332E 39 N297S/I332E 40 P230A/E233D/I332E 41P244H/P245A/P247V 42 S239D/A330L/I332E 43 S239D/A330Y/I332E 44S239D/A330Y/I332E/K326E 45 S239D/A330Y/I332E/K326T 46S239D/A330Y/I332E/L234I 47 S239D/A330Y/I332E/L235D 48S239D/A330Y/I332E/V240I 49 S239D/A330Y/I332E/V264T 50S239D/A330Y/I332E/V266I 51 S239D/D265F/N297D/I332E 52S239D/D265H/N297D/I332E 53 S239D/D265I/N297D/I332E 54S239D/D265L/N297D/I332E 55 S239D/D265T/N297D/I332E 56S239D/D265V/N297D/I332E 57 S239D/D265Y/N297D/I332E 58 S239D/I332D 59S239D/I332E 60 S239D/I332E/A330I 61 S239D/I332N 62 S239D/I332Q 63S239D/N297D/I332E 64 S239D/N297D/I332E/A330Y 65 S239D/N297D/I332E/A330Y/F241S/F243H/V262T/V264T 66 S239D/N297D/I332E/K326E 67S239D/N297D/I332E/L235D 68 S239D/S298A/I332E 69 S239D/V264I/A330L/I332E70 S239D/V264I/I332E 71 S239D/V264I/S298A/I332E 72 S239E/D265N 73S239E/D265Q 74 S239E/I332D 75 S239E/I332E 76 S239E/I332N 77 S239E/I332Q78 S239E/N297D/I332E 79 S239E/V264I/A330Y/I332E 80 S239E/V264I/I332 E 81S239E/V264I/S298A/A330Y/ I332E 82 S239N/A330L/I332E 83 S239N/A330Y/I332E84 S239N/I332D 85 S239N/I332E 86 S239N/I332N 87 S239N/I332Q 88S239N1S298A/I332E 89 S239Q/I332D 90 S239Q/I332E 91 S239Q/I332N 92S239Q/I332Q 93 S239Q/V264I/I332E 94 S298A/I332E 95 V264E/N297D/I332E 96V264I/A330L/I332E 97 V264I/A330Y/I332E 98 V264I/I332E 99V264I/S298A/I332E 100 Y296D/N297D/I332E 101 Y296E/N297D/I332 E 102Y296H/N297D/I332E 103 Y296N/N297D/I332E 104 Y296Q/N297I/I332E 105Y296T/N297D/I332E.

Effector function can be modified by techniques such as those describedin the Antibody Engineering Technology Art, or by other means. Forexample, cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region, resultingin the generation of a homodimeric antibody that may have improvedinternalization capability and/or increased complement-mediated cellkilling and ADCC. See Caron et al. (1992) J. Exp Med. 176:1191-1195; andB. Shopes (1992) J. Immunol. 148:2918-2922. Homodimeric antibodies withenhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. (1993)Cancer Research 53:2560-2565. Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. Stevenson et al. (1989)Anti-Cancer Drug Design 3:219-230.

The affinities and binding properties of the molecules of the inventionfor an FcγR are initially determined using in vitro assays (biochemicalor immunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to ELISA assay, surface plasmon resonanceassay, immunoprecipitation assays (See Section 6.2.2). Preferably, thebinding properties of the molecules of the invention are alsocharacterized by in vitro functional assays for determining one or moreFcγR mediator effector cell functions (See Section 6.2.2). In mostpreferred embodiments, the molecules of the invention have similarbinding properties in in vivo models (such as those described anddisclosed herein) as those in in vitro based assays. However, thepresent invention does not exclude molecules of the invention that donot exhibit the desired phenotype in in vitro based assays but doexhibit the desired phenotype in vivo.

In some embodiments, the molecules of the invention comprising a variantFc region comprise at least one amino acid modification in the CH3domain of the Fc region, which is defined as extending from amino acids342-447. In other embodiments, the molecules of the invention comprisinga variant Fc region comprise at least one amino acid modification in theCH2 domain of the Fc region, which is defined as extending from aminoacids 231-341. In some embodiments, the molecules of the inventioncomprise at least two amino acid modifications, wherein one modificationis in the CH3 region and one modification is in the CH2 region. Theinvention further encompasses amino acid modification in the hingeregion. Molecules of the invention with one or more amino acidmodifications in the CH₂ and/or CH3 domains have altered affinities foran FcγR as determined using methods described herein or known to oneskilled in the art.

In a particular embodiment, the invention encompasses amino acidmodification in the CH1 domain of the Fc region.

In particularly preferred embodiments, the invention encompassesmolecules comprising a variant Fc region wherein said variant has anincreased binding to FcγRIIA (CD32A) and/or an increased ADCC activity,as measured using methods known to one skilled in the art andexemplified herein. The ADCC assays used in accordance with the methodsof the invention may be NK dependent or macrophage dependent.

The Fc variants of the present invention may be combined with otherknown Fc modifications including but not limited to modifications whichalter effector function and modification which alter FcγR bindingaffinity. In a particular embodiment, an Fc variant of the inventioncomprising a first amino acid modification in the CH3 domain. CH2 domainor the hinge region may be combined with a second Fc modification suchthat the second Fc modification is not in the same domain as the firstso that the first Fc modification confers an additive, synergistic ornovel property on the second Fc modification. In some embodiments, theFc variants of the invention do not have any amino acid modification inthe CH2 domain.

In a preferred specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule has an altered affinity for an FcγR, providedthat said variant Fc region does not have a substitution at positionsthat make a direct contact with FcγR based on crystallographic andstructural analysis of Fc-FcγR interactions such as those disclosed bySondermann et al., 2000 (Nature, 406: 267-273 which is incorporatedherein by reference in its entirety). Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(hinge region), amino acids 265-269 (B/C loop), amino acids 297-299(C′/E loop), and amino acids 327-332 (F/G) loop. In some embodiments,the molecules of the invention comprising variant Fc regions comprisemodification of at least one residue that makes a direct contact with anFcγR based on structural and crystallographic analysis.

The FcγR interacting domain maps to the lower hinge region and selectsites within the CH2 and CH3 domains of the IgG heavy chain. Amino acidresidues flanking the actual contact positions and amino acid residuesin the CH3 domain play a role in IgG/FcγR interactions as indicated bymutagenesis studies and studies using small peptide inhibitors,respectively (Sondermann et al., 2000 Nature, 406: 267-273; Diesenhoferet al., 1981, Biochemistry, 20: 2361-2370; Shields et al., 2001, J.Biol. Chem. 276: 6591-6604; each of which is incorporated herein byreference in its entirety). Direct contact as used herein refers tothose amino acids that are within at least 1 A, at least 2, or at least3 angstroms of each other or within 1 Å, 1.2 Å, 1.5 Å, 1.7 Å or 2 Å VanDer Waals radius.

In another preferred embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule binds an FcγR via the Fc region with an alteredaffinity relative to a molecule comprising a wild-type Fc region,provided that said variant Fc region does not have or is not solely asubstitution at any of positions 255, 256, 258, 267, 268, 269, 270, 272,276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298,300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331,333, 334, 335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430,434, 435, 437, 438, 439. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said molecule binds an FcγRvia the Fc region with an altered affinity relative to a moleculecomprising a wild-type Fc region, provided that said variant Fc regiondoes not have or is not solely a substitution at any of positions 255,258, 267, 269, 270, 276, 278, 280, 283, 285, 289, 292, 293, 294, 295,296, 300, 303, 305, 307, 309, 322, 329, 332, 331, 337, 338, 340, 373,376, 416, 419, 434, 435, 437, 438, 439 and does not have an alanine atany of positions 256, 290, 298, 312, 333, 334, 359, 360 326, or 430; alysine at position 330; a threonine at position 339; a methionine atposition 320; a serine at position 326; an asparagine at position 326;an aspartic acid at position 326; a glutamic acid at position 326; aglutamine at position 334; a glutamic acid at position 334; a methionineat position 334; a histidine at position 334; a valine at position 334;or a leucine at position 334; a lysine at position 335 an asparagine atposition 268; a glutamine at position 272; a glutamine, serine, oraspartic acid at position 286; a serine at position 290; a methionine,glutamine, glutamic acid, or arginine at position 320; a glutamic acidat position 322; a serine, glutamic acid, or aspartic acid at position326; a lysine at position 330; a glutamine at position 335; or amethionine at position 301.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region does nothave or is not solely a substitution at any of positions 268, 269, 270,272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309,331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434,435, 437, 438 or 439 and does not have a histidine, glutamine, ortyrosine at position 280; a serine, glycine, threonine or tyrosine atposition 290, a leucine or isoleucine at position 300; an asparagine atposition 294, a proline at position 296; a proline, asparagine, asparticacid, or valine at position 298; a lysine at position 295. In yetanother preferred embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule binds an FcγR via the Fc region with a reducedaffinity relative to a molecule comprising a wild-type Fc regionprovided that said variant Fc region does not have or is not solely asubstitution at any of positions 252, 254, 265, 268, 269, 270, 278, 289,292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333,335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437,438, or 439. In yet another preferred embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said molecule binds an FcγRvia the Fc region with an enhanced affinity relative to a moleculecomprising a wild-type Fc region provided that said variant Fc regiondoes not have or is not solely a substitution at any of positions 280,283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315,331, 333, 334, 337, 340, 360, 378, 398, or 430.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region does notinclude or are not solely a substitution at any of positions 330, 243,247, 298, 241, 240, 244, 263, 262, 235, 269, or 328 and does not have aleucine at position 243, an asparagine at position 298, a leucine atposition 241, and isoleucine or an alanine at position 240, a histidineat position 244, a valine at position 330, or an isoleucine at position328.

In most preferred embodiments, the molecules of the invention withaltered affinities for activating and/or inhibitory receptors havingvariant Fc regions, have one or more amino acid modifications, whereinsaid one or more amino acid modification is a substitution at position288 with asparagine, at position 330 with serine and at position 396with leucine (MgFc10); or a substitution at position 334 with glutamicacid, at position 359 with asparagine, and at position 366 with serine(MgFc13); or a substitution at position 316 with aspartic acid, atposition 378 with valine, and at position 399 with glutamic acid(MgFc27); or a substitution at position 392 with threonine, and atposition 396 with leucine (MgFc38); or a substitution at position 221with glutamic acid, at position 270 with glutamic acid, at position 308with alanine, at position 311 with histidine, at position 396 withleucine, and at position 402 with aspartic acid (MgFc42); or asubstitution at position 240 with alanine, and at position 396 withleucine (MgFc52); or a substitution at position 410 with histidine, andat position 396 with leucine (MgFc53); or a substitution at position 243with leucine, at position 305 with isoleucine, at position 378 withaspartic acid, at position 404 with serine, and at position 396 withleucine (MgFc54); or a substitution at position 255 with isoleucine, andat position 396 with leucine (MgFc55); or a substitution at position 370with glutamic acid and at position 396 with leucine (MgFc59); or asubstitution at position 243 with leucine, at position 292 with proline,at position 300 with leucine, at position 305 with isoleucine, and atposition 396 with leucine (MgFc88); or a substitution at position 243with leucine, at position 292 with proline, at position 300 withleucine, and at position 396 with leucine (MgFc88A); or a substitutionat position 234 with leucine, at position 292 with proline, and atposition 300 with leucine (MgFc155); or a substitution at position 243with leucine, at position 292 with proline, and at position 300 withleucine; or a substitution at position 243 with leucine, at position 292with proline, and at position 396 with leucine; or a substitution atposition 243 with leucine, and at position 292 with proline; or asubstitution at position 243 with leucine; or a substitution at position273 with phenylalanine; or a substitution at position 247 with leucine,at position 270 with glutamic acid, and at position 421 with lysine.

In one specific embodiment, the invention encompasses a moleculecomprising a variant Fc region wherein said variant Fc region comprisesa substitution at position 396 with leucine, at position 270 withglutamic acid and at position 243 with leucine. In another specificembodiment the molecule further comprises one or more amino acidmodification such as those disclosed herein.

In some embodiments, the invention encompasses molecules comprising avariant Fc region having an amino acid modification at one or more ofthe following positions: 119, 125, 132, 133, 141, 142, 147, 149, 162,166, 185, 192, 202, 205, 210, 214, 217, 219, 215, 216, 217, 218, 219,221, 222, 223, 224, 225, 227, 288, 229, 231, 232, 233, 234, 235, 240,241, 242, 243, 244, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256,258, 261, 262, 263, 268, 269, 270, 272, 273, 274, 275, 276, 279, 280,281, 282, 284, 287, 288, 289, 290, 291, 292, 293, 295, 298, 300, 301,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 315, 316, 317,318, 319, 320, 323, 326, 327, 328, 330, 333, 334, 335, 337, 339, 340,343, 344, 345, 347, 348, 352, 353, 354, 355, 358, 359, 360, 361, 362,365, 366, 367, 369, 370, 371, 372, 375, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 404, 406, 407, 408, 409, 410, 411, 412, 414,415, 416417, 419, 420, 421, 422, 423, 424, 427, 428, 431, 433, 435, 436,438, 440, 441, 442, 443, 446, 447. Preferably such mutations result inmolecules that have an altered affinity for an FcγR and/or have analtered effector cell mediated function as determined using methodsdisclosed and exemplified herein and known to one skilled in the art.

In some embodiments, the molecules, preferably the immunoglobulins ofthe invention further comprise one or more glycosylation sites, so thatone or more carbohydrate moieties are covalently attached to themolecule. Preferably, the antibodies of the invention with one or moreglycosylation sites and/or one or more modifications in the Fc regionhave an enhanced antibody mediated effector function, e.g., enhancedADCC activity. In some embodiments, the invention further comprisesantibodies comprising one or more modifications of amino acids that aredirectly or indirectly known to interact with a carbohydrate moiety ofthe antibody, including but not limited to amino acids at positions 241,243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and301. Amino acids that directly or indirectly interact with acarbohydrate moiety of an antibody are known in the art, see, e.g.,Jefferis et al., 1995 Immunology Letters, 44: 111-7, which isincorporated herein by reference in its entirety.

Glycosylation is a co-translational/post-translational modification thatresults in the attachment of a glycosylated high mannose oligosaccharide(GlcNAc2Man9Glu3), which is subsequently processed, first to aGlcNAc₂Man₉ (“Man9”) structure (FIG. 41), and then successively toproduce a GlcNAc₂Man₈ (“Man8”), a GlcNAc₂Man₇ (“Man7”), a GlcNAc₂Man₆(“Man6”) and ultimately, a GlcNAc₂Man₅ (“Man5”) structure. TheGlcNAc₂Man₅ (“Man5”) structure is then processed by the successiveaction of glycosyltransferases to generate a “G0F,” “G1F” and “G2F”oligosaccharide that exhibits a complex diantennary structure (FIG. 42)(Jefferis, R. (2005) “Glycosylation of Recombinant AntibodyTherapeutics,” Biotechnol. Prog. 21:11-16; Kornfeld, R. et al. (1985)“Assembly Of Asparagine-Linked Oligosaccharides,” Ann. Rev. Biochem.54:631-664).

The invention encompasses antibodies that have been modified byintroducing one or more glycosylation sites into one or more sites ofthe antibodies, preferably without altering the functionality of theantibody, e.g., binding activity to FcγR. Glycosylation sites may beintroduced into the variable and/or constant region of the antibodies ofthe invention. As used herein, “glycosylation sites” include anyspecific amino acid sequence in an antibody to which an oligosaccharide(i.e., carbohydrates containing two or more simple sugars linkedtogether) will specifically and covalently attach. Oligosaccharide sidechains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of anoligosaccharide moiety to the side chain of an asparagine residue.O-linked glycosylation refers to the attachment of an oligosaccharidemoiety to a hydroxyamino acid, e.g., serine, threonine. The antibodiesof the invention may comprise one or more glycosylation sites, includingN-linked and O-linked glycosylation sites. Any glycosylation site forN-linked or O-linked glycosylation known in the art may be used inaccordance with the instant invention. An exemplary N-linkedglycosylation site that is useful in accordance with the methods of thepresent invention, is the amino acid sequence: Asn-X-Thr/Ser, wherein Xmay be any amino acid and Thr/Ser indicates a threonine or a serine.Such a site or sites may be introduced into an antibody of the inventionusing methods well known in the art to which this invention pertains.See, for example, “In Vitro Mutagenesis,” Recombinant DNA: A ShortCourse, J. D. Watson, et al. W.H. Freeman and Company, New York, 1983,chapter 8, pp. 106-116, which is incorporated herein by reference in itsentirety. An exemplary method for introducing a glycosylation site intoan antibody of the invention may comprise: modifying or mutating anamino acid sequence of the antibody so that the desired Asn-X-Thr/Sersequence is obtained.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all ofwhich are incorporated herein by reference in their entirety. In otherembodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by deleting one ormore endogenous carbohydrate moieties of the antibody. In a specificembodiment, the invention encompasses shifting the glycosylation site ofthe Fc region of an antibody, by modifying position 297 (e.g., fromasparagine to a residue without an available amine group, e.g.,glutamine) and/or positions adjacent to 297. In a specific embodiment,the invention encompasses modifying position 296 so that position 296and not position 297 is glycosylated.

Fucosylated carbohydrate structures are involved in a variety ofbiological and pathological processes in eukaryotic organisms includingtissue development, angiogenesis, fertilization, cell adhesion,inflammation, and tumor metastasis (Ma, B. et al. (Epub 2006 Sep. 14)“Fucosylation In Prokaryotes And Eukaryotes,” Glycobiology.16(12):158R-184R). Therapeutic antibodies fully lacking the core fucoseof the Fc oligosaccharides have been found to exhibit much higher ADCCin humans than their fucosylated counterparts (Iida, S. et al. (2009)“Two Mechanisms Of The Enhanced Antibody-Dependent Cellular Cytotoxicity(ADCC) Efficacy Of Non-Fucosylated Therapeutic Antibodies In HumanBlood,” BMC Cancer. 18:9:58). However, the production of such antibodieshas previously required the enzymatic removal of fucose residues (forexample, using N-glycosidase F) that had been added to the antibodies bya glycosylase.

One aspect of the present invention relates to the recognition thatvariations in the Fc region of an antibody can interfere with thecellular glycosylation mechanism and thereby yield antibodies thatexhibit a decreased extent of glycosylation (and in particular, offucosylation).

The invention thus provides a means for making antibodies having adecreased extent of glycosylation (and in particular, of fucosylation)by substituting one, or more two, three, four, five or more of thenative Fc residues to form a variant Fc region. Without intending to bebound by any theory of mechanism of action, it is believed that suitablesubstitutions yield a polypeptide having a variant Fc region that iseither less fucosylated than a native Fc region, or is not fucosylatedat all, and that such polypeptides, due to their altered (or absent)extent of glycosylation (and in particular, of fucosylation) exhibitimproved effector function, relative to polypeptides having a native Fcregion. Polynucleotides encoding polypeptides that comprise such variantFc regions can therefore be introduced into host cells (e.g., CHO cells,yeast, etc.), including host cells that are capable of mediating normalglycosylation (and in particular, normal fucosylation), but will beexpressed as polypeptides that are less post-translationally modified byglycosylase (or fucosylase) or are not post-translationally modified bysuch enzymes, relative to polypeptides comprising native Fc regions andthereby exhibit improved effector function (e.g., improved binding tothe activating receptors (e.g., CD16A, CD32A) and reduced binding toCD32B. Such improvement of effector function may be measured using anoff-rate analysis. Such analysis provides the best indicator forpotential improvement of in vivo FcγR binding activity because as akinetic variable it directly reflects the Fc-FcγR interactionindependent of antibody concentration.

As used herein, such decreased extent of glycosylation (and inparticular, of fucosylation) is preferably less than 80%, morepreferably less than 60%, still more preferably less than 40%, and mostpreferably less than 20% of the extent of glycosylation (and inparticular, of fucosylation) exhibited by such antibody on the absenceof such variation in its Fc region. In a more preferred embodiment, suchvariations in the Fc region of the antibody will substantially eliminateor fully eliminate the extent of glycosylation (and in particular, offucosylation) exhibited by such antibody. The ability of such Fcvariants to decrease the extent of glycosylation (and in particular, offucosylation) is a general characteristic and is exemplified herein withrespect to anti-Her2/neu antibodies.

The invention particularly concerns polypeptides (e.g., antibodies) thatpossess variant Fc regions that result in an enhanced ratio of highmannose oligosaccharide glycosylation to complex oligosaccharideglycosylation. As used herein, such a ratio denotes a comparison of theextent to which the glycosylation exhibited by an Fc region is a complexoligosaccharide of either G0F, G1F or G2F (see, FIG. 42) relative to theextent to which the glycosylation exhibited by an Fc region is Man5,Man6, Man7, Man8 or Man9 (see, FIG. 41). The ratio of high mannoseoligosaccharide glycosylation to complex oligosaccharide glycosylationis therefore:Σ (% Man5+% Man6+% Man7+% Man8+% Man9): Σ (% G0F+% G1F+% G2F).Preferably, the enhanced ratio of high mannose oligosaccharideglycosylation to complex oligosaccharide glycosylation will be greaterthan about 0.2. More preferably, this ratio will be greater than about0.5, still more preferably this ratio will be greater than about 1.0,greater than about 1.5, greater than about 2.0, greater than about 2.5,greater than about 3.0, greater than about 3.5, greater than about 4.0,greater than about 4.5, greater than about 5.0, greater than about 5.5,or greater than 6.0. Most preferably, the upper ranges of such ratiowill be less than about 10, more preferably, less than about 9.5, lessthan about 9, less than about 8.5, less than about 8, less than about7.5, less than about 7, less than about 6.5, less than about 6, or lessthan about 5.5. Specific contemplated ranges of such ratio includegreater than about 0.2 but less than about 5.5; greater than about 0.5but less than about 5.5; greater than about 1.0 but less than about 5.5;greater than about 2.0 but less than about 5.5; greater than about 3.0but less than about 5.5; greater than about 4.0 but less than about 5.5;and greater than about 5.0 but less than about 5.5.

Preferably, Fc variants exhibiting a decreased extent of glycosylation(and in particular, of fucosylation) comprise an amino acid substitutionof either or both of positions L234 and/or L235. More preferably, suchFc variants will further comprise at least one additional amino acidsubstitution at any or all of positions: F243, R292, Y300, V305, and/orP396. Thus, preferred Fc variants exhibiting a decreased extent ofglycosylation (and in particular, of fucosylation) comprise amino acidsubstitutions at either or both of positions L234 and/or L235, and alsoat position F243; position R292; position Y300; position V305; positionP396; positions F243 and R292; positions F243 and Y300; positions F243and V305; positions F243 and P396; positions R292 and Y300; positionsR292 and V305; positions R292 and P396; positions Y300 and V305;positions Y300 and P396; positions V305 and P396; positions F243, R292and Y300; positions F243, R292 and V305; positions F243, R292 and P396;positions F243, Y300 and V305; positions F243, Y300 and P396; positionsF243, V305 and P396; positions R292, Y300 and V305, positions R292, Y300and P396; positions R292, V305 and P396; positions Y300, V305 and P396;positions F243, R292, Y300 and V305; positions F243, R292, Y300 andP396; positions F243, R292, V305 and P396; positions F243, Y300, V305and P396; positions R292, Y300, V305 and P396; or positions F243, R292,Y300, V305 and P396. The L234 substitutions L234F and/or the L235substitution L235V are particularly preferred.

6.1 Polypeptides and Antibodies with Variant Fc Regions

It will be appreciated by one skilled in the art that aside from aminoacid substitutions, the present invention contemplates othermodifications of the Fc region amino acid sequence in order to generatean Fc region variant with one or more altered properties, e.g., alteredeffector function. The invention contemplates deletion of one or moreamino acid residues of the Fc region in order to reduce binding to anFcγR. Preferably, no more than 5, no more than 10, no more than 20, nomore than 30, no more than 50 Fc region residues will be deletedaccording to this embodiment of the invention. The Fc region hereincomprising one or more amino acid deletions will preferably retain atleast about 80%, and preferably at least about 90%, and most preferablyat least about 95%, of the wild type Fc region. In some embodiments, oneor more properties of the molecules are maintained such as for example,non-immunogenicity, FcγRIIIA binding, FcγRIIA binding, or a combinationof these properties.

In alternate embodiments, the invention encompasses amino acid insertionto generate the Fc region variants, which variants have alteredproperties including altered effector function. In one specificembodiment, the invention encompasses introducing at least one aminoacid residue, for example one to two amino acid residues and preferablyno more than 10 amino acid residues adjacent to one or more of the Fcregion positions identified herein. In alternate embodiments, theinvention further encompasses introducing at least one amino acidresidue, for example one to two amino acid residues and preferably nomore than 10 amino acid residues adjacent to one or more of the Fcregion positions known in the art as impacting FcγR interaction and/orbinding.

The invention further encompasses incorporation of unnatural amino acidsto generate the Fc variants of the invention. Such methods are known tothose skilled in the art such as those using the natural biosyntheticmachinery to allow incorporation of unnatural amino acids into proteins,see, e.g., Wang et al., 2002 Chem. Comm. 1:1-11; Wang et al., 2001,Science, 292: 498-500; van Hest et al., 2001. Chem. Comm. 19: 1897-1904,each of which is incorporated herein by reference in its entirety.Alternative strategies focus on the enzymes responsible for thebiosynthesis of amino acyl-tRNA, see, e.g., Tang et al., 2001, J. Am.Chem. 123(44): 11089-11090; Kiick et al., 2001, FEBS Lett. 505(3): 465;each of which is incorporated herein by reference in its entirety.

The affinities and binding properties of the Fc variants, or fragmentsthereof, of use in the invention are initially determined using ayeast-display system, preferably combined with in vitro assays(biochemical or immunological based assays) known in the art fordetermining Fc-FcγR interactions, i.e., specific binding of an Fc regionto an FcγR including but not limited to ELISA assay, surface plasmonresonance assay, immunoprecipitation assays (See Section 6.2.1). Incertain embodiments, candidate Fc variants identified using the yeastdisplay system are further incorporated into an antibody or fragmentthereof for testing in said in vitro assay. Preferably, the bindingproperties of the molecules of the invention are also characterized byin vitro functional assays for determining one or more FcγR mediatoreffector cell functions (See Section 6.2.5). Such methods havepreviously been disclosed by the inventors, see, e.g., U.S PatentApplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351, each of whichis hereby incorporated by reference in its entirety, and have been usedto identify and characterize novel Fc mutations based on bindingcharacteristics to FcγRIIIA and FcγRIIB, see, e.g., Table 5.

TABLE 5 Fc MUTATIONS IDENTIFIED USING YEAST DISPLAY AND ELISA ASSAY IIIAIIB Clone # Mutation sites Domain binding binding 4 A339V, Q347H CH2,CH3 + + 5 L251P, S415I CH2, CH3 + + 8 V185M, K218N, R292L, D399E CH1,hinge, CH2, no change − CH3 12 K290E, L142P CH1, CH2 + not tested 16A141V, H268L, K288E, P291S CH1, CH2 − not tested 19 L133M, P150Y, K205E,S383N, CH1, CH2, CH3 − not tested N384K 21 P396L CH3 • •+ 25 P396H CH3••• •• 6 K392R CH3 no change no change 15 R301C, M252L, S192T CH1, CH2 −not tested 17 N315I CH2 no change not tested 18 S132I CH1 no change nottested 26 A162V CH1 no change not tested 27 V348M, K334N, F275I, Y202M,CH1, Ch2 + + K147T 29 H310Y, T289A, G337E CH2 − not tested 30 S119F,G371S, Y407N, E258D CH1, CH2, CH3 + no change 31 K409R, S166N CH1, CH3no change not tested 20 S408I, V215I, V125I CH1, Hinge, CH3 + no change24 G385E, P247H CH2, CH3 ••• + 16 V379M CH3 •• no change 17 S219Y Hinge• − 18 V282M CH2 • − 31 F275I, K334N, V348M CH2 + no change 35 D401VCH3 + no change 37 V280L, P395S CH2 + − 40 K222N Hinge • no change 41K246T, Y319F CH2 • no change 42 F243I, V379L CH2, CH3 •+ − 43 K334E CH2•+ − 44 K246T, P396H CH2, CH3 • ••+ 45 H268D, E318D CH2 •+ ••••• 49K288N, A330S, P396L CH2, CH3 ••••• ••• 50 F243L, R255L, E318K CH2 • − 53K334E, T359N, T366S CH2, CH3 • no change 54 I377F CH3 •+ + 57 K334I CH2• no change 58 P244H, L358M, V379M, CH2, CH3 •+ •+ N384K, V397M 59K334E, T359N, T366S CH2, CH3 •+ no change (independent isolate) 61 I377F(independent isolate) CH3 ••• ••+ 62 P247L CH2 •• ••+ 64 P217S, A378V,S408R Hinge, CH3 •• ••••+ 65 P247L, I253N, K334N CH2 ••• ••+ 66 K288M,K334E CH2 ••• − 67 K334E, E380D CH2, CH3 •+ − 68 P247L (independentisolate) CH2 + •••• 69 T256S, V305I, K334E, N390S CH2, CH3 •+ no change70 K326E CH2 •+ ••+ 71 F372Y CH3 + •••••+ 72 K326E (independent isolate)CH2 + •• 74 K334E, T359N, T366S CH2, CH3 •• no change (independentisolate) 75 K334E (independent isolate) CH2 ••+ no change 76 P396L(independent isolate) CH3 •+ no change 78 K326E (independent isolate)CH2 •• •••+ 79 K246I, K334N CH2 • •••• 80 K334E (independent isolate)CH2 • no change 81 T335N, K370E, A378, T394M, CH2, CH3 • no change S424L82 K320E, K326E CH2 • • 84 H224L Hinge • ••••• 87 S375C, P396L CH3 •+••••+ 89 E233D, K334E CH2 •+ no change 91 K334E (independent isolate)CH2 • no change 92 K334E (independent isolate) CH2 • no change 94 K334E,T359N, T366S, Q386R CH2 • no change relative to comparable molecule withwild-type Fc region, • ≡ 1-fold increase in affinity; + ≡ up to 50%increase in affinity; and − ≡ 1-fold decrease in affinity

In most preferred embodiments, the molecules of the invention havesimilar binding properties in in vivo models (such as those disclosedherein and/or known in the art) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo. A representativeflow chart of the screening and characterization of molecules of theinvention is described in FIG. 1.

The invention encompasses molecules comprising a variant Fc region thatbinds with a greater affinity to one or more FcγRs. Such moleculespreferably mediate effector function more effectively as discussedinfra. In other embodiments, the invention encompasses moleculescomprising a variant Fc region that bind with a weaker affinity to oneor more FcγRs. Reduction or elimination of effector function isdesirable in certain cases for example in the case of antibodies whosemechanism of action involves blocking or antagonism but not killing ofthe cells bearing a target antigen. Reduction or elimination of effectorfunction would be desirable in cases of autoimmune disease where onewould block FcγR activating receptors in effector cells (This type offunction would be present in the host cells). In general increasedeffector function would be directed to tumor and foreign cells.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that altereffector function. The invention encompasses combining an Fc variant ofthe invention with other Fc modifications to provide additive,synergistic, or novel properties in antibodies or Fc fusions. Preferablythe Fc variants of the invention enhance the phenotype of themodification with which they are combined. For example, if an Fc variantof the invention is combined with a mutant known to bind FcγRIIIA with ahigher affinity than a comparable molecule comprising a wild type Fcregion; the combination with a mutant of the invention results in agreater fold enhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:49634969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, JImmunol 164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573;U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of whichis incorporated herein by reference in its entirety.

In some embodiments, the Fc variants of the present invention areincorporated into an antibody or Fc fusion that comprises one or moreengineered glycoforms, i.e., a carbohydrate composition that iscovalently attached to a molecule comprising an Fc region, wherein saidcarbohydrate composition differs chemically from that of a parentmolecule comprising an Fc region. Engineered glycoforms may be usefulfor a variety of purposes, including but not limited to enhancing orreducing effector function. Engineered glycoforms may be generated byany method known to one skilled in the art, for example by usingengineered or variant expression strains, by co-expression with one ormore enzymes, for example DI N-acetylglucosaminyltransferase III(GnTI11), by expressing a molecule comprising an Fc region in variousorganisms or cell lines from various organisms, or by modifyingcarbohydrate(s) after the molecule comprising Fc region has beenexpressed. Methods for generating engineered glycoforms are known in theart, and include but are not limited to those described in Umana et al,1999. Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawaet al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S.Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylationengineering technology (GLYCART biotechnology AG, Zurich, Switzerland);each of which is incorporated herein by reference in its entirety. See,e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al., 2004,JMB, 336: 1239-49 each of which is incorporated herein by reference inits entirety.

The Fc variants of the present invention may be optimized for a varietyof properties. Properties that may be optimized include but are notlimited to enhanced or reduced affinity for an FcγR, enhanced or reducedeffector function. In a preferred embodiment, the Fc variants of thepresent invention are optimized to possess enhanced affinity for a humanactivating FcγR, preferably FcγR, FcγRIIA, FcγRIIc, FcγRIIIA, andFcγRIIIB, most preferably FcγRIIIA. In an alternate preferredembodiment, the Fc variants are optimized to possess reduced affinityfor the human inhibitory receptor FcγRIIB. These preferred embodimentsare anticipated to provide antibodies and Fc fusions with enhancedtherapeutic properties in humans, for example enhanced effector functionand greater anti-cancer potency as described and exemplified herein.These preferred embodiments are anticipated to provide antibodies and Fcfusions with enhanced tumor elimination in mouse xenograft tumor models.

In an alternate embodiment the Fc variants of the present invention areoptimized to have reduced affinity for a human FcγR, including but notlimited to FcγRI, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, and FcγRIIIB.These embodiments are anticipated to provide antibodies and Fc fusionswith enhanced therapeutic properties in humans, for example reducedeffector function and reduced toxicity.

In alternate embodiments the Fc variants of the present inventionpossess enhanced or reduced affinity for FcγRs from non-human organisms,including but not limited to mice, rats, rabbits, and monkeys. Fcvariants that are optimized for binding to a non-human FcγR may find usein experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing ofantibodies or Fc fusions that comprise Fc variants that are optimizedfor one or more mouse FcγRs, may provide valuable information withregard to the efficacy of the antibody or Fc fusion, its mechanism ofaction, and the like. In certain embodiments, molecules of the inventioncomprising a variant human Fc region are tested in transgenic miceexpressing one or more human Fcγ receptors (e.g., FcγRIIIA, FcγRIIA,FcγRIIB).

While it is preferred to alter binding to an FcγR, the instant inventionfurther contemplates Fc variants with altered binding affinity to theneonatal receptor (FcRn). Although not intending to be bound by aparticular mechanism of action, Fc region variants with improvedaffinity for FcRn are anticipated to have longer serum half-lives, andsuch molecules will have useful applications in methods of treatingmammals where long half-life of the administered polypeptide is desired,e.g., to treat a chronic disease or disorder. Although not intending tobe bound by a particular mechanism of action, Fc region variants withdecreased FcRn binding affinity, on the contrary, are expected to haveshorter half-lives, and such molecules may, for example, be administeredto a mammal where a shortened circulation time may be advantageous,e.g., for in vivo diagnostic imaging or for polypeptides which havetoxic side effects when left circulating in the blood stream forextended periods. Fc region variants with decreased FcRn bindingaffinity are anticipated to be less likely to cross the placenta, andthus may be utilized in the treatment of diseases or disorders inpregnant women.

In other embodiments, these variants may be combined with other known Fcmodifications with altered FcRn affinity such as those disclosed inInternational Publication Nos. WO 98/23289; and WO 97/34631; and U.S.Pat. No. 6,277,375, each of which is incorporated herein by reference inits entirety.

The invention encompasses any other method known in the art forgenerating antibodies having an increased half-life in vivo, forexample, by introducing one or more amino acid modifications (i.e.,substitutions, insertions or deletions) into an IgG constant domain, orFcRn binding fragment thereof (preferably a Fc or hinge-Fc domainfragment). See, e.g., International Publication Nos. WO 98/23289; and WO97/34631; and U.S. Pat. No. 6,277,375, each of which is incorporatedherein by reference in its entirety to be used in combination with theFc variants of the invention. Further, antibodies of the invention canbe conjugated to albumin in order to make the antibody or antibodyfragment more stable in vivo or have a longer half-life in vivo. Thetechniques well-known in the art, see, e.g., International PublicationNos. WO 93/15199, WO 93/15200, and WO 01/77137, and European Patent No.EP 413,622, all of which are incorporated herein by reference in theirentirety.

The variant(s) described herein may be subjected to furthermodifications, often times depending on the intended use of the variant.Such modifications may involve further alteration of the amino acidsequence (substitution, insertion and/or deletion of amino acidresidues), fusion to heterologous polypeptide(s) and/or covalentmodifications. Such further modifications may be made prior to,simultaneously with, or following, the amino acid modification(s)disclosed herein which results in altered properties such as analteration of Fc receptor binding and/or ADCC activity.

Alternatively or additionally, the invention encompasses combining theamino acid modifications disclosed herein with one or more further aminoacid modifications that alter C1q binding and/or complement dependentcytoxicity function of the Fc region as determined in vitro and/or invivo. Preferably, the starting molecule of particular interest herein isusually one that binds to C1q and displays complement dependentcytotoxicity (CDC). The further amino acid substitutions describedherein will generally serve to alter the ability of the startingmolecule to bind to C1q and/or modify its complement dependentcytotoxicity function, e.g., to reduce and preferably abolish theseeffector functions. In other embodiments molecules comprisingsubstitutions at one or more of the described positions with improvedC1q binding and/or complement dependent cytotoxicity (CDC) function arecontemplated herein. For example, the starting molecule may be unable tobind C1q and/or mediate CDC and may be modified according to theteachings herein such that it acquires these further effector functions.Moreover, molecules with preexisting C1q binding activity, optionallyfurther having the ability to mediate CDC may be modified such that oneor both of these activities are altered, e.g., enhanced. In someembodiments, the invention encompasses variant Fc regions with alteredCDC activity without any alteration in C1q binding. In yet otherembodiments, the invention encompasses variant Fc regions with alteredCDC activity and altered C1q binding.

To generate an Fc region with altered C1q binding and/or complementdependent cytotoxicity (CDC) function, the amino acid positions to bemodified are generally selected from positions 270, 322, 326, 327, 329,331, 333, and 334, where the numbering of the residues in an IgG heavychain is that of the EU index as in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (199). These amino acidmodifications may be combined with one or more Fc modificationsdisclosed herein to provide a synergistic or additive effect on C1qbinding and/or CDC activity. In other embodiments, the inventionencompasses Fc variants with altered C1q binding and/or complementdependent cytotoxicity (CDC) function comprising an amino acidsubstitution at position 396 with leucine and at position 255 withleucine; or an amino acid substitution at position 396 with leucine andat position 419 with histidine; an amino acid substitution at position396 with leucine and at position 370 with glutamic acid; an amino acidsubstitution at position 396 with leucine and at position 240 withalanine; an amino acid substitution at position 396 with leucine and atposition 392 with threonine; an amino acid substitution at position 247with leucine and at position 421 with lysine. The invention encompassesany known modification of the Fc region which alters C1q binding and/orcomplement dependent cytotoxicity (CDC) function such as those disclosedin Idusogie et al., 2001, J. Immunol. 166(4) 2571-5; Idusogie et al., J.Immunol. 2000 164(8): 4178-4184; each of which is incorporated herein byreference in its entirety.

As disclosed above, the invention encompasses an Fc region with alteredeffector function, e.g., modified C1q binding and/or FcR binding andthereby altered CDC activity and/or ADCC activity. In specificembodiments, the invention encompasses variant Fc regions with improvedC1q binding and improved FcγRIII binding; e.g. having both improved ADCCactivity and improved CDC activity. In alternative embodiments, theinvention encompasses a variant Fc region with reduced CDC activityand/or reduced ADCC activity. In other embodiments, one may increaseonly one of these activities, and optionally also reduce the otheractivity, e.g. to generate an Fc region variant with improved ADCCactivity, but reduced CDC activity and vice versa.

6.1.1 Mutants with Enhanced Altered Affinities for FcγRIIIA and/orFcγRIIA

The invention encompasses molecules comprising a variant Fc region,having one or more amino acid modifications (e.g., substitutions) in oneor more regions, wherein such modifications alter the affinity of thevariant Fc region for an activating FcγR. In some embodiments, moleculesof the invention comprise a variant Fc region, having one or more aminoacid modifications (e.g., substitutions) in one or more regions, whichmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA by at least 2-fold, relative to a comparablemolecule comprising a wild-type Fc region. In another specificembodiment, molecules of the invention comprise a variant Fc region,having one or more amino acid modifications (e.g., substitutions) in oneor more regions, which modifications increase the affinity of thevariant Fc region for FcγRIIIA and/or FcγRIIA by greater than 2 fold,relative to a comparable molecule comprising a wild-type Fc region. Inother embodiments of the invention the one or more amino acidmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA by at least 3-fold, 4-fold, 5-fold, 6-fold,8-fold, or 10-fold relative to a comparable molecule comprising awild-type Fc region. In yet other embodiments of the invention the oneor more amino acid modifications decrease the affinity of the variant Fcregion for FcγRIIIA and/or FcγRIIA by at least 3-fold, 4-fold, 5-fold,6-fold, 8-fold, or 10-fold relative to a comparable molecule comprisinga wild-type Fc region. Such fold increases are preferably determined byan ELISA or surface plasmon resonance assays. In a specific embodiment,the one or more amino acid modifications do not include or are notsolely a substitution at any one of positions 329, 331, or 322 with anyamino acid. In certain embodiments, the one or more amino acidmodifications do not include or are not solely a substitution with anyone of alanine at positions 256, 290, 298, 312, 333, 334, 359, 360, or430; with lysine at position 330; with threonine at position 339; withmethionine at position 320; with serine, asparagine, aspartic acid, orglutamic acid at position 326 with glutamine, glutamic acid, methionine,histidine, valine, or leucine at position 334. In another specificembodiment, the one or more amino acid modifications do not include orare not solely a substitution at any of positions 280, 290, 300, 294, or295. In another more specific embodiment, the one or more amino acidmodifications do not include or are not solely a substitution atposition 300 with leucine or isoleucine; at position 295 with lysine; atposition 294 with asparagine; at position 298 with aspartic acid,proline, aspargine, or valine; at position 280 with histidine, glutamineor tyrosine; at position 290 with serine, glycine, thionine or tyrosine.

In another specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIA via it Fc regionwith a greater affinity than a comparable molecule comprising thewild-type Fc region binds FcγRIIA, provided that said variant Fc regiondoes not have an alanine at any of positions 256, 290, 326, 255, 258,267, 272, 276, 280, 283, 285, 286, 331, 337, 268, 272, or 430; anasparagine at position 268; a glutamine at position 272; a glutamine,serine, or aspartic acid at position 286; a serine at position 290; amethionine, glutamine, glutamic acid, or arginine at position 320; aglutamic acid at position 322; a serine, glutamic acid, or aspartic acidat position 326; a lysine at position 330; a glutamine at position 335;or a methionine at position 301. In a specific embodiment, molecules ofthe invention comprise a variant Fc region, having one or more aminoacid modifications (e.g., substitutions) in one or more regions, whichmodifications increase the affinity of the variant Fc region for FcγRIIAby at least 2-fold, relative to a comparable molecule comprising awild-type Fc region. In another specific embodiment, molecules of theinvention comprise a variant Fc region, having one or more amino acidmodifications (e.g., substitutions) in one or more regions, whichmodifications increase the affinity of the variant Fc region for FcγRIIAby greater than 2 fold, relative to a comparable molecule comprising awild-type Fc region. In other embodiments of the invention the one ormore amino acid modifications increase the affinity of the variant Fcregion for FcγRIIA by at least 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,or 10-fold relative to a comparable molecule comprising a wild-type Fcregion

In a specific embodiment, the invention encompasses molecules,preferably polypeptides, and more preferably immunoglobulins (e.g.,antibodies), comprising a variant Fc region, having one or more aminoacid modifications (e.g., substitutions but also include insertions ordeletions), which modifications increase the affinity of the variant Fcregion for FcγRIIIA and/or FcγRIIA by at least 65%, at least 70%, atleast 75%, at least 85%, at least 90%, at least 95%, at least 99%, atleast 100%, at least 150%, and at least 200%, relative to a comparablemolecule comprising a wild-type Fc region.

In a specific embodiment, the one or more amino acid modifications whichincrease the affinity of the variant Fc region for one or moreactivating FcγRs comprise a substitution at position 347 with histidine,and at position 339 with valine; or a substitution at position 425 withisoleucine and at position 215 with phenylalanine; or a substitution atposition 408 with isoleucine, at position 215 with isoleucine, and atposition 125 with leucine; or a substitution at position 385 withglutamic acid and at position 247 with histidine; or a substitution atposition 348 with methionine, at position 334 with asparagine, atposition 275 with isoleucine, at position 202 with methionine, and atposition 147 with threonine; or a substitution at position 275 withisoleucine, at position 334 with asparagine, and at position 348 withmethionine; or a substitution at position 279 with leucine and atposition 395 with serine; or a substitution at position 246 withthreonine and at position 319 with phenylalanine; or a substitution atposition 243 with isoleucine and at position 379 with leucine; or asubstitution at position 243 with leucine, at position 255 with leucineand at position 318 with lysine; or a substitution at position 334 withglutamic acid, at position 359 with asparagine, and at position 366 withserine; or a substitution at position 288 with methionine and atposition 334 with glutamic acid; or a substitution at position 334 withglutamic acid and at position 380 with aspartic acid; or a substitutionat position 256 with serine, at position 305 with isoleucine, atposition 334 with glutamic acid and at position 390 with serine; or asubstitution at position 335 with asparagine, at position 370 withglutamic acid, at position 378 with valine, at position 394 withmethionine, and at position 424 with leucine; or a substitution atposition 233 with aspartic acid and at position 334 with glutamic acid;or a substitution at position 334 with glutamic acid, at position 359with asparagine, at position 366 with serine, and at position 386 witharginine; or a substitution at position 246 with threonine and atposition 396 with histidine; or a substitution at position 268 withaspartic acid and at position 318 with aspartic acid; or a substitutionat position 288 with asparagine, at position 330 with serine, and atposition 396 with leucine; or a substitution at position 244 withhistidine, at position 358 with methionine, at position 379 withmethionine, at position 384 with lysine and at position 397 withmethionine; or a substitution at position 217 with serine, at position378 with valine, and at position 408 with arginine; or a substitution atposition 247 with leucine, at position 253 with asparagine, and atposition 334 with asparagine; or a substitution at position 246 withisoleucine, and at position 334 with asparagine; or a substitution atposition 320 with glutamic acid and at position 326 with glutamic acid;or a substitution at position 375 with cysteine and at position 396 withleucine; or a substitution at position 243 with leucine, at position 292with proline, at position 300 with leucine, at position 305 withisoleucine, and at position 396 with leucine; or a substitution atposition 243 with leucine, at position 292 with proline, at position 300with leucine, and at position 396 with leucine; or a substitution atposition 234 with leucine, at position 292 with proline, and at position300 with leucine; or a substitution at position 234 with leucine, atposition 292 with proline, and at position 396 with leucine; or asubstitution at position 234 with leucine, at position 292 with proline,and at position 305 with isoleucine; or a substitution at position 234with leucine and at position 292 with proline; or a substitution atposition 234 with leucine; or a substitution at position 247 withleucine, at position 270 with glutamic acid, and at position 421 withlysine. Examples of other amino acid substitutions that result in anenhanced affinity for FcγRIIIA in vitro are disclosed below andsummarized in Table 3.

The invention encompasses a molecule comprising a variant Fc region,wherein said variant Fc region comprises a substitution at position 243with isoleucine and at position 379 with leucine, such that saidmolecule binds FcγRIIIA with about a 1.5 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 288 withasparagine, at position 330 with serine, and at position 396 withleucine, such that said molecule binds FcγRIIIA with about a 5 foldhigher affinity than a comparable molecule comprising the wild type Fcregion binds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 243 with leucine and at position 255 with leucine such thatsaid molecule binds FcγRIIIA with about a 1 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 334 with glutamicacid, at position 359 with asparagine, and at position 366 with serine,such that said molecule binds FcγRIIIA with about a 1.5 fold higheraffinity than a comparable molecule comprising the wild type Fc regionbinds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 288 with methionine and at position 334 with glutamic acid,such that said molecule binds FcγRIIIA with about a 3 fold higheraffinity than a comparable molecule comprising the wild type Fc regionbinds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 316 with aspartic acid, at position 378 with valine, and atposition 399 with glutamic acid, such that said molecule binds FcγRIIIAwith about a 1.5 fold higher affinity than a comparable moleculecomprising the wild type Fc region binds FcγRIIIA, as determined by anELISA assay. In a specific embodiment, the invention encompasses amolecule comprising a variant Fc region, wherein said variant Fc regioncomprises a substitution at position 315 with isoleucine, at position379 with methionine, and at position 399 with glutamic acid, such thatsaid molecule binds FcγRIIIA with about a 1 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 243 withisoleucine, at position 379 with leucine, and at position 420 withvaline, such that said molecule binds FcγRIIIA with about a 2.5 foldhigher affinity than a comparable molecule comprising the wild type Fcregion binds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 247 with leucine, and at position 421 with lysine, such thatsaid molecule binds FcγRIIIA with about a 3 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 392 withthreonine and at position 396 with leucine such that said molecule bindsFcγRIIIA with about a 4.5 fold higher affinity than a comparablemolecule comprising the wild type Fc region binds FcγRIIIA, asdetermined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 293 with valine,at position 295 with glutamic acid, and at position 327 with threonine,such that said molecule binds FcγRIIIA with about a 1.5 fold higheraffinity than a comparable molecule comprising the wild type Fc regionbinds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 268 with asparagine and at position 396 with leucine, such thatsaid molecule binds FcγRIIIA with about a 2 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 319 withphenylalanine, at position 352 with leucine, and at position 396 withleucine, such that said molecule binds FcγRIIIA with about a 2 foldhigher affinity than a comparable molecule comprising the wild type Fcregion binds FcγRIIIA, as determined by an ELISA assay.

In a specific embodiment, the invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with a greater affinity than a comparable polypeptidecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 396 with histidine. In aspecific embodiment, the invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 248 with methionine. The invention encompassesan isolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a similar affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 392 witharginine. The invention encompasses an isolated polypeptide comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIIA with a similar affinitythan a comparable polypeptide comprising the wild-type Fc region,wherein said at least one amino acid modification comprises substitutionat position 315 with isoleucine. The invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with a similar affinity than a comparable polypeptidecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 132 with isoleucine. Theinvention encompasses an isolated polypeptide comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a similar affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position162 with valine. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 396 with leucine. The invention encompasses anisolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 379 withmethionine. The invention encompasses an isolated polypeptide comprisinga variant Fc region, wherein said variant Fc region comprises at leastone amino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIIA with a greater affinitythan a comparable polypeptide comprising the wild-type Fc region,wherein said at least one amino acid modification comprises substitutionat position 219 with tyrosine. The invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with a greater affinity than a comparable polypeptidecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 282 with methionine. Theinvention encompasses an isolated polypeptide comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a greater affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position401 with valine. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 222 with asparagine. The invention encompassesan isolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 334 withglutamic acid. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 377 with phenylalanine. The inventionencompasses an isolated polypeptide comprising a variant Fc region,wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a greater affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position334 with isoleucine. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 247 with leucine. The invention encompasses anisolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 326 withglutamic acid. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 372 with tyrosine. The invention encompasses anisolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 224 withleucine.

The invention encompasses an isolated polypeptide comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a greater affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position275 with tyrosine. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 398 with valine. The invention encompasses anisolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 334 withasparagine. The invention encompasses an isolated polypeptide comprisinga variant Fc region, wherein said variant Fc region comprises at leastone amino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIIA with a greater affinitythan a comparable polypeptide comprising the wild-type Fc region,wherein said at least one amino acid modification comprises substitutionat position 400 with proline. The invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with a greater affinity than a comparable polypeptidecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 407 with isoleucine. Theinvention encompasses an isolated polypeptide comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a greater affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position372 with tyrosine. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a similaraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 366 with asparagine. The invention encompassesan isolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a reduced affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 414 withasparagine. The invention encompasses an isolated polypeptide comprisinga variant Fc region, wherein said variant Fc region comprises at leastone amino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIIA with a reduced affinitythan a comparable polypeptide comprising the wild-type Fc region,wherein said at least one amino acid modification comprises substitutionat position 225 with serine. The invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with a reduced affinity than a comparable polypeptidecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 377 with asparagine. Theinvention encompasses an isolated polypeptide comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a greater affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position243 with leucine. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with a greateraffinity than a comparable polypeptide comprising the wild-type Fcregion, wherein said at least one amino acid modification comprisessubstitution at position 292 with proline. The invention encompasses anisolated polypeptide comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 300 withleucine. The invention encompasses an isolated polypeptide comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIIA with a greater affinitythan a comparable polypeptide comprising the wild-type Fc region,wherein said at least one amino acid modification comprises substitutionat position 305 with isoleucine. The invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with a greater affinity than a comparable polypeptidecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 396 with leucine. Theinvention encompasses an isolated polypeptide comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIIA with a greater affinity than acomparable polypeptide comprising the wild-type Fc region, wherein saidat least one amino acid modification comprises substitution at position273 with phenylalanine.

In a specific embodiment, the invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIIA with about a 2 fold greater affinity than a comparablepolypeptide comprising the wild-type Fc region as determined by an ELISAassay, wherein said at least one amino acid modification comprisessubstitution at position 379 with methionine. In another specificembodiment, the invention encompasses an isolated poly peptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIIA with about a 1.5fold greater affinity than a comparable polypeptide comprising thewild-type Fc region as determined by an ELISA assay, wherein said atleast one amino acid modification comprises substitution at position 248with methionine.

In some embodiments, the molecules of the invention have an alteredaffinity for FcγRIIIA and/or FcγRIIA as determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining Fc-FcγR interactions, i.e., specific binding of an Fc regionto an FcγR including but not limited to ELISA assay, surface plasmonresonance assay, immunoprecipitation assays (See Section 6.2.5.1).Preferably, the binding properties of these molecules with alteredaffinities for activating FcγR receptors are also correlated to theiractivity as determined by in vitro functional assays for determining oneor more FcγR mediator effector cell functions (See Section 6.2.7), e.g.,molecules with variant Fc regions with enhanced affinity for FcγRIIIAhave an enhanced ADCC activity. In most preferred embodiments, themolecules of the invention that have an altered binding property for anactivating Fc receptor, e.g., FcγRIIIA in an in vitro assay also have analtered binding property in in vivo models (such as those described anddisclosed herein). However, the present invention does not excludemolecules of the invention that do not exhibit an altered FcγR bindingin in vitro based assays but do exhibit the desired phenotype in vivo.

6.1.2 Mutants with Enhanced Affinity for FcγRIIIA and Reduced or noAffinity for FcγRIIB

In a specific embodiment, the molecules of the invention comprise avariant Fc region, having one or more amino acid modifications (i.e.,substitutions) in one or more regions, which one or more modificationsincrease the affinity of the variant Fc region for FcγRIIIA anddecreases the affinity of the variant Fc region for FcγRIIB, relative toa comparable molecule comprising a wild-type Fc region which bindsFcγRIIIA and FcγRIIB with wild-type affinity. In a certain embodiment,the one or more amino acid modifications do not include or are notsolely a substitution with alanine at any of positions 256, 298, 333,334, 280, 290, 294, 298, or 296; or a substitution at position 298 withasparagine, valine, aspartic acid, or proline; or a substitution 290with serine. In certain amino embodiments, the one or more amino acidmodifications increases the affinity of the variant Fc region forFcγRIIIA by at least 65%, at least 70%, at least 75%, at least 85%, atleast 90%, at least 95%, at least 99%, at least 100%, at least 200%, atleast 300%, at feat 400% and decreases the affinity of the variant Fcregion for FcγRIIB by at least 65%, at least 70%, at least 75%, at least85%, at least 90%, at least 95%, at least 99%, at least 100%, at least200%, at least 300%, at least 400%.

In a specific embodiment, the molecule of the invention comprising avariant Fc region with an enhanced affinity for FcγRIIIA and a loweredaffinity or no affinity for FcγRIIB, as determined based on an ELISAassay and/or an ADCC based assay using ch-4-4-20 antibody, or a surfaceplasmon resonance assay using a chimeric 4D5 antibody, carrying thevariant Fc region comprises a substitution at position 275 withisoleucine, at position 334 with asparagine, and at position 348 withmethionine; or a substitution at position 279 with leucine and atposition 395 with serine; or a substitution at position 246 withthreonine and at position 319 with phenylalanine; or a substitution atposition 243 with leucine, at position 255 with leucine, and at position318 with lysine; or a substitution at position 334 with glutamic acid,at position 359 with asparagine and at position 366 with serine; or asubstitution at position 334 with glutamic acid and at position 380 withaspartic acid; or a substitution at position 256 with serine, atposition 305 with isoleucine, at position 334 with glutamic acid, and atposition 390 with serine; or a substitution at position 335 withasparagine, at position 370 with glutamic acid, at position 378 withvaline, at position 394 with methionine and at position 424 withleucine; or a substitution at position 233 with aspartic acid and atposition 334 with glutamic acid; or a substitution at position 334 withglutamic acid, at position 359 with asparagine, at position 366 withserine and at position 386 with arginine; or a substitution at position312 with glutamic acid, at position 327 with asparagine, and at position378 with serine; or a substitution at position 288 with asparagine andat position 326 with asparagine; or a substitution at position 247 withleucine and at position 421 with lysine; or a substitution at position298 with asparagine and at position 381 with arginine; or a substitutionat position 280 with glutamic acid, at position 354 with phenylalanine,at position 431 with aspartic acid, and at position 441 with isoleucine;or a substitution at position 255 with glutamine and at position 326with glutamic acid; or a substitution at position 218 with arginine, atposition 281 with aspartic acid and at position 385 with arginine; or asubstitution at position 247 with leucine, at position 330 withthreonine and at position 440 with glycine; or a substitution atposition 284 with alanine and at position 372 with leucine; or asubstitution at position 335 with asparagine, as position 387 withserine and at position 435 with glutamine; or a substitution at position247 with leucine, at position 431 with valine and at position 442 withphenylalanine; or a substitution at position 243 with leucine, atposition 292 with proline, at position 305 with isoleucine, and atposition 396 with leucine; or a substitution at position 243 leucine, atposition 292 with proline, and at position 305 with isoleucine; or asubstitution at position 292 with proline, at position 305 withisoleucine, and at position 396 with leucine; or a substitution atposition 243 with leucine, and at position 292 with proline; or asubstitution at position 292 with proline; or a substitution at position243 with leucine, at position 292 with proline, and at position 396 withleucine; or a substitution at position 243 with leucine, at position 292with proline, at position 300 with leucine; or a substitution atposition 243 with leucine.

In a specific embodiment, the molecule of the invention comprising avariant Fc region with an enhanced affinity for FcγRIIIA and a loweredaffinity or no affinity for FcγRIIB as determined based on an ELISAassay and/or an ADCC based assay using ch-4-4-20 antibody carrying thevariant Fc region comprises a substitution at position 379 withmethionine; at position 219 with tyrosine; at position 282 withmethionine; at position 401 with valine; at position 222 withasparagine; at position 334 with isoleucine; at position 334 withglutamic acid; at position 275 with tyrosine; at position 398 withvaline. In yet another specific embodiment, the molecule of theinvention comprising a variant Fc region with an enhanced affinity forFcγRIIIA and a lowered affinity or no affinity for FcγRIIB as determinedbased on an ELISA assay and/or an ADCC based assay using ch-4-4-20antibody, or a surface plasmon resonance assay using a chimeric 4D5antibody, carrying the variant Fc region comprises a substitution atposition 243 with leucine; at position 292 with proline; and at position300 with leucine.

The invention encompasses an isolated polypeptide comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIB with about a 3 fold loweraffinity than a comparable polypeptide comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 288 withasparagine, at position 330 with serine, and at position 396 withleucine. The invention encompasses an isolated polypeptide comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIB with about a 10-15 foldlower affinity than a comparable polypeptide comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 316 with asparticacid, at position 378 with valine, and at position 399 with glutamicacid. The invention encompasses an isolated polypeptide comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIB with about a 10 fold loweraffinity than a comparable polypeptide comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 315 withisoleucine, at position 379 with methionine, and at position 399 withglutamic acid. The invention encompasses an isolated polypeptidecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIB with about a 7fold lower affinity than a comparable polypeptide comprising thewild-type Fc region as determined by an ELISA assay, wherein said atleast one amino acid modification comprises substitution at position 243with isoleucine, at position 379 with leucine, and at position 420 withvaline. The invention encompasses an isolated polypeptide comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid polypeptide specifically binds FcγRIIB with about a 3 fold loweraffinity than a comparable polypeptide comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 392 with threonineand at position 396 with leucine. The invention encompasses an isolatedpolypeptide comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said polypeptide specifically bindsFcγRIIB with about a 5 fold lower affinity than a comparable polypeptidecomprising the wild-type Fc region as determined by an ELISA assay,wherein said at least one amino acid modification comprises substitutionat position 268 with asparagine and at position 396 with leucine. Theinvention also encompasses an isolated polypeptide comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidpolypeptide specifically binds FcγRIIB with about a 2 fold loweraffinity than a comparable polypeptide comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 319 withphenylalanine, at position 352 with leucine, and at position 396 withleucine.

6.1.3 Mutants with Enhanced Affinity to FcγRIIIA and FcγRIIB

The invention encompasses molecules comprising variant Fc regions,having one or more amino acid modifications, which modificationsincrease the affinity of the variant Fc region for FcγRIIIA and FcγRIIBby at least 65%, at least 70%, at least 75%, at least 85%, at least 90%,at least 95%, at least 99%, at least 100%, at least 200%, at least 300%,at least 400% and decreases the affinity of the variant Fc region forFcγRIIB by at least 65%, at least 70%, at least 75%, at least 85%, atleast 90%, at least 95%, at least 99%, at least 100%, at least 200%, atleast 300%, at least 400%. In a specific embodiment, the molecule of theinvention comprising a variant Fc region with an enhanced affinity forFcγRIIIA and an enhanced affinity for FcγRIIB (as determined based on anELISA assay and/or an ADCC based assay using ch-4-4-20 antibody, or asurface plasmon resonance assay using a chimeric 4D5 antibody, carryingthe variant Fc region as described herein) comprises a substitution atposition 415 with isoleucine and at position 251 with phenylalanine; ora substitution at position 399 with glutamic acid, at position 292 withleucine, and at position 185 with methionine; or a substitution atposition 408 with isoleucine, at position 215 with isoleucine, and atposition 125 with leucine; or a substitution at position 385 withglutamic acid and at position 247 with histidine; or a substitution atposition 348 with methionine, at position 334 with asparagine, atposition 275 with isoleucine, at position 202 with methionine and atposition 147 with threonine; or a substitution at position 246 withthreonine and at position 396 with histidine; or a substitution atposition 268 with aspartic acid and at position 318 with aspartic acid;or a substitution at position 288 with asparagine, at position 330 withserine and at position 396 with leucine; or a substitution at position244 with histidine, at position 358 with methionine, at position 379with methionine, at position 384 with lysine and at position 397 withmethionine; or a substitution at position 217 with serine, at position378 with valine, and at position 408 with arginine; or a substitution atposition 247 with leucine, at position 253 with asparagine, and atposition 334 with asparagine; or a substitution at position 246 withisoleucine and at position 334 with asparagine; or a substitution atposition 320 with glutamic acid and at position 326 with glutamic acid;or a substitution at position 375 with cysteine and at position 396 withleucine; or a substitution at position 343 with serine, at position 353with leucine, at position 375 with isoleucine, at position 383 withasparagine; or a substitution at position 394 with methionine and atposition 397 with methionine; or a substitution at position 216 withaspartic acid, at position 345 with lysine and at position 375 withisoleucine; or a substitution at position 288 with asparagine, atposition 330 with serine, and at position 396 with leucine; or asubstitution at position 247 with leucine and at position 389 withglycine; or a substitution at position 222 with asparagine, at position335 with asparagine, at position 370 with glutamic acid, at position 378with valine and at position 394 with methionine; or a substitution atposition 316 with aspartic acid, at position 378 with valine and atposition 399 with glutamic acid; or a substitution at position 315 withisoleucine, at position 379 with methionine, and at position 394 withmethionine; or a substitution at position 290 with threonine and atposition 371 with aspartic acid; or a substitution at position 247 withleucine and at position 398 with glutamine; or a substitution atposition 326 with glutamine; at position 334 with glutamic acid, atposition 359 with asparagine, and at position 366 with serine; or asubstitution at position 247 with leucine and at position 377 withphenylalanine; or a substitution at position 378 with valine, atposition 390 with isoleucine and at position 422 with isoleucine; or asubstitution at position 326 with glutamic acid and at position 385 withglutamic acid; or a substitution at position 282 with glutamic acid, atposition 369 with isoleucine and at position 406 with phenylalanine; ora substitution at position 397 with methionine; at position 411 withalanine and at position 415 with asparagine; or a substitution atposition 223 with isoleucine, at position 256 with serine and atposition 406 with phenylalanine; or a substitution at position 298 withasparagine and at position 407 with arginine; or a substitution atposition 246 with arginine, at position 298 with asparagine, and atposition 377 with phenylalanine; or a substitution at position 235 withproline, at position 382 with methionine, at position 304 with glycine,at position 305 with isoleucine, and at position 323 with isoleucine; ora substitution at position 247 with leucine, at position 313 witharginine, and at position 388 with glycine; or a substitution atposition 221 with tyrosine, at position 252 with isoleucine, at position330 with glycine, at position 339 with threonine, at position 359 withasparagine, at position 422 with isoleucine, and at position 433 withleucine; or a substitution at position 258 with aspartic acid, and atposition 384 with lysine; or a substitution at position 241 with leucineand at position 258 with glycine; or a substitution at position 370 withasparagine and at position 440 with asparagine; or a substitution atposition 317 with asparagine and a deletion at position 423; or asubstitution at position 243 with isoleucine, at position 379 withleucine and at position 420 with valine; or a substitution at position227 with serine and at position 290 with glutamic acid; or asubstitution at position 231 with valine, at position 386 withhistidine, and at position 412 with methionine; or a substitution atposition 215 with proline, at position 274 with asparagine, at position287 with glycine, at position 334 with asparagine, at position 365 withvaline and at position 396 with leucine; or a substitution at position293 with valine, at position 295 with glutamic acid and at position 327with threonine; or a substitution at position 319 with phenylalanine, atposition 352 with leucine, and at position 396 with leucine; or asubstitution at position 392 with threonine and at position 396 withleucine; at a substitution at position 268 with asparagine and atposition 396 with leucine; or a substitution at position 290 withthreonine, at position 390 with isoleucine, and at position 396 withleucine; or a substitution at position 326 with isoleucine and atposition 396 with leucine; or a substitution at position 268 withaspartic acid and at position 396 with leucine; or a substitution atposition 210 with methionine and at position 396 with leucine; or asubstitution at position 358 with proline and at position 396 withleucine; or a substitution at position 288 with arginine, at position307 with alanine, at position 344 with glutamic acid, and at position396 with leucine; or a substitution at position 273 with isoleucine, atposition 326 with glutamic acid, at position 328 with isoleucine and atposition 396 with leucine; or a substitution at position 326 withisoleucine, at position 408 with asparagine and at position 396 withleucine; or a substitution at position 334 with asparagine and atposition 396 with leucine; or a substitution at position 379 withmethionine and at position 396 with leucine; or a substitution atposition 227 with serine and at position 396 with leucine; or asubstitution at position 217 with serine and at position 396 withleucine; or a substitution at position 261 with asparagine, at position210 with methionine and at position 396 with leucine; or a substitutionat position 419 with histidine and at position 396 with leucine; or asubstitution at position 370 with glutamic acid and at position 396 withleucine; or a substitution at position 242 with phenylalanine and atposition 396 with leucine; or a substitution at position 255 withleucine and at position 396 with leucine: or a substitution at position240 with alanine and at position 396 with leucine; or a substitution atposition 250 with serine and at position 396 with leucine; or asubstitution at position 247 with serine and at position 396 withleucine; or a substitution at position 410 with histidine and atposition 396 with leucine; or a substitution at position 419 withleucine and at position 396 with leucine; or a substitution at position427 with alanine and at position 396 with leucine; or a substitution atposition 258 with aspartic acid and at position 396 with leucine; or asubstitution at position 384 with lysine and at position 396 withleucine; or a substitution at position 323 with isoleucine and atposition 396 with leucine; or a substitution at position 244 withhistidine and at position 396 with leucine; or a substitution atposition 305 with leucine and at position 396 with leucine; or asubstitution at position 400 with phenylalanine and at position 396 withleucine; or a substitution at position 303 with isoleucine and atposition 396 with leucine; or a substitution at position 243 withleucine, at position 305 with isoleucine, at position 378 with asparticacid, at position 404 with serine and at position 396 with leucine; or asubstitution at position 290 with glutamic acid, at position 369 withalanine, at position 393 with alanine and at position 396 with leucine;or a substitution at position 210 with asparagine, at position 222 withisoleucine, at position 320 with methionine and at position 396 withleucine; or a substitution at position 217 with serine, at position 305with isoleucine, at position 309 with leucine, at position 390 withhistidine and at position 396 with leucine; or a substitution atposition 246 with asparagine; at position 419 with arginine and atposition 396 with leucine; or a substitution at position 217 withalanine, at position 359 with alanine and at position 396 with leucine;or a substitution at position 215 with isoleucine, at position 290 withvaline and at position 396 with leucine; or a substitution at position275 with leucine, at position 362 with histidine, at position 384 withlysine and at position 396 with leucine; or a substitution at position334 with asparagine; or a substitution at position 400 with proline; ora substitution at position 407 with isoleucine; or a substitution atposition 372 with tyrosine; or a substitution at position 366 withasparagine; or a substitution at position 414 with asparagine; or asubstitution at position 352 with leucine; or a substitution at position225 with serine; or a substitution at position 377 with asparagine; or asubstitution at position 248 with methionine; or a substitution atposition 243 with leucine, at position 292 with proline, at position 300with leucine, at position 305 with isoleucine, and at position 396 withleucine; or a substitution at position 243 with leucine, at position 292with proline, at position 300 with leucine, and at position 396 withleucine; or a substitution at position 243 with leucine, and at position396 with leucine; or at position 292 with proline, and at position 305with isoleucine.

6.1.4 Mutants that do not Bind any FcγR

In some embodiments, the invention encompasses molecules comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, which variantFc region does not bind any FcγR, as determined by standard assays knownin the art and disclosed herein, relative to a comparable moleculecomprising the wild type Fc region. In a specific embodiment, the one ormore amino acid modifications which abolish binding to all FcγRscomprise a substitution at position 232 with serine and at position 304with glycine; or a substitution at position 269 with lysine, at position290 with asparagine, at position 311 with arginine, and at position 433with tyrosine; or a substitution at position 252 with leucine; or asubstitution at position 216 with aspartic acid, at position 334 witharginine, and at position 375 with isoleucine; or a substitution atposition 247 with leucine and at position 406 with phenylalanine, or asubstitution at position 335 with asparagine, at position 387 withserine, and at position 435 with glutamine; or a substitution atposition 334 with glutamic acid, at position 380 with aspartic acid, andat position 446 with valine; or a substitution at position 303 withisoleucine, at position 369 with phenylalanine, and at position 428 withleucine; or a substitution at position 251 with phenylalanine and atposition 372 with leucine; or a substitution at position 246 withglutamic acid, at position 284 with methionine and at position 308 withalanine; or a substitution at position 399 with glutamic acid and atposition 402 with aspartic acid; or a substitution at position 399 withglutamic acid and at position 428 with leucine.

6.1.5 Mutants with Altered FcγR-Mediated Effector Functions

The invention encompasses immunoglobulin comprising Fc variants withaltered effector functions. In some embodiments, immunoglobulinscomprising Fc variants mediate effector function more effectively in thepresence of effector cells as determined using assays known in the artand exemplified herein. In other embodiments, immunoglobulins comprisingFc variants mediate effector function less effectively in the presenceof effector cells as determined using assays known in the art andexemplified herein. In specific embodiments, the Fc variants of theinvention may be combined with other known Fc modifications that altereffector function, such that the combination has an additive,synergistic effect. The Fc variants of the invention have alteredeffector function in vitro and/or in vivo.

In a specific embodiment, the immunoglobulins of the invention withenhanced affinity for FcγRIIIA and/or FcγRIIA have an enhancedFcγR-mediated effector function as determined using ADCC activity assaysdisclosed herein. Examples of effector functions that could be mediatedby the molecules of the invention include, but are not limited to, C1qbinding, complement-dependent cytotoxicity, antibody-dependent cellmediate cytotoxicity (ADCC), phagocytosis, etc. The effector functionsof the molecules of the invention can be assayed using standard methodsknown in the art, examples of which are disclosed in Section 6.2.6.

In a specific embodiment, the immunoglobulins of the inventioncomprising a variant Fc region with enhanced affinity for FcγRIIIAand/or FcγRIIA mediate antibody dependent cell mediated cytotoxicity(ADCC) 2-fold more effectively, than an immunoglobulin comprising awild-type Fc region. In other embodiments, the immunoglobulins of theinvention comprising a variant Fc region with enhanced affinity forFcγRIIIA and/or FcγRIIA mediate antibody dependent cell mediatedcytotoxicity (ADCC) at least 4-fold, at least 8-fold, at least 10-fold,at least 100-fold, at least 1000-fold, at least 10⁴-fold, at least10⁵-fold more effectively, than an immunoglobulin comprising a wild-typeFc region. In another specific embodiment, the immunoglobulins of theinvention with enhanced affinity for FcγRIIIA and/or FcγRIIA havealtered C1q binding activity. In some embodiments, the immunoglobulinsof the invention with enhanced affinity for FcγRIIIA and/or FcγRIIA haveat least 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, atleast 100-fold, at least 1000-fold, at least 10⁴-fold, at least 10⁵-foldhigher C1q binding activity than an immunoglobulin comprising awild-type Fc region. In yet another specific embodiment, theimmunoglobulins of the invention with enhanced affinity for FcγRIIIAand/or FcγRIIA have altered complement dependent cytotoxicity. In yetanother specific embodiment, the immunoglobulins of the invention withenhanced affinity for FcγRIIIA and/or FcγRIIA have an enhancedcomplement dependent cytotoxicity than an immunoglobulin comprising awild-type Fc region. In some embodiments, the immunoglobulins of theinvention with enhanced affinity for FcγRIIIA and/or FcγRIIA have atleast 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, atleast 100-fold, at least 1000-fold, at least 10⁴-fold, at least 10⁵-foldhigher complement dependent cytotoxicity than an immunoglobulincomprising a wild-type Fc region.

In certain embodiments, the Fc variants of the invention may be combinedwith or comprise any of the Fc variants previously identified by theinventors to modulate effector function as disclosed in U.S PatentApplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351, each of whichis hereby incorporated by reference in its entirety. Examples of such Fcvariants previously identified by the authors are provided in Tables 6and 7 infra.

In other embodiments, immunoglobulins of the invention with enhancedaffinity for FcγRIIIA and/or FcγRIIA have enhanced phagocytosis activityrelative to an immunoglobulin comprising a wild-type Fc region, asdetermined by standard assays known to one skilled in the art ordisclosed herein. In some embodiments, the immunoglobulins of theinvention with enhanced affinity for FcγRIIIA and/or FcγRIIA have atleast 2-fold, at least 4-fold, at least 8-fold, at least 10-fold higherphagocytosis activity relative to an immunoglobulin comprising awild-type Fc region.

TABLE 6 Summary of ADCC Activity of Mutants in ch4D5 ADCC Fc Variant 1μg/ml 0.5 μg/ml Amino Acid % Specific % Specific Label Ref VariationLysis Normalized Lysis Normalized MGFc-27 2C4 G316D, A378V, 33% 2.24 22%3.60 D399E MGFc-31 3B9 P247L, N421K 30% 2.05 17% 2.90 MGFc-10 1E1 K288N,A330S, 24% 1.66 10% 1.67 P396L MGFc-28 2C5 N315I, V379M, 20% 1.37 10%1.69 T394M MGFc-29 3D11 F243I, V379L, 20% 1.35 7% 1.17 G420V CH4-4-20(P54008) 15% 1.00 6 1.00 MGFc-35 3D2 R255Q, K326E 11% 0.79 3% 0.53MGFc-36 3D3 K218R, G281D, 10% 0.67 5% 0.78 G385R MGFc-30 3A8 F275Y 9%0.64 2% 0.37 MGFc-32 3C8 D280E, S354F, 9% 0.62 4% 0.75 A431D, L441IMGFc-33 3C9 K317N, F423Δ 3% 0.18 −1% −0.22 MGFc-34 3B10 F421L, E258G −1%−0.08 −4% −0.71 MGFc-26 D265A 1% 0.08 −3% −0.45

TABLE 7 SUMMARY OF MUTANTS ELISA ELISA 4-4-20 Anti-HER2 Fc Amino AcidFcR3A, FcR2B, IIIA IIB Phagocytosis ADCC ADCC Variant changesK_(D)/K_(off) K_(D)/K_(off) binding binding (mutant/WT) (mutant/wt)(mutant/wt) Wt none 198/0.170 94/.094 1 1    1 1 1 MGFc 5 V379M160/0.167 70/0.10 2X N/C 0.86 2.09 1.77 MGFc 9 P243I, V379L 99.7/0.105 120/0.113 1.5X reduced ? 2.25 2.04 MGFc 10 K288N, A330S, 128/0.11533.4/0.050  5X 3X 1.2 2.96 2.50 P396L MGFc 11 F243L, R255L  90/0.07574.7/0.09  1x reduced 0.8 2.38 1.00 MGFc13 K334E, T359N, 55.20.12872/0.11 1.5X N/C [ 1.57 3.67 T366S MGFc 14 K288M, K334E 75.4/0.1  95.6/0.089 3X reduced [ 1.74 MGFc 23 K334E, R292L 70.2/0.105  108/0.107[ 2.09 1.6 MGFc 27 G316D, A378V,  72/0.117 46/0.06 1.5X 14X  1.4 3.606.88 D399E MGFc 28 N315I, A379M, 1X 9X 1.37 1.69 1.00 D399E MGFc 29P243I, V379L, 108/0.082 93.4/.101   2.5X 7X 0.93 1.17 1.00 G420V MGFc 31P247L, N421K  62/0.108  66/0.065 3X N/C 1.35 2.90 1.00 MGFc 37 K248M154/0.175 100/0.091 1.4X reduced 0.98 3.83 0.67 MGFc 38 K392T, P396L 84/0.104  50/0.041 4.5X   2.5X 1.4 3.07 2.50 MGFc 39 E293V, Q295E,195/0.198  86/0.074 1.4X reduced 1.5 4.29 0.50 A327T MGFc 40 K248M180/0.186 110/0.09  1.4X reduced 1.14 4.03 MGFc 41 H268N, P396L178/0.159 46.6/0.036  2.2X   4.5X 1.96 2.24 0.67 MGFc 43 Y319F, P352L,125/0.139 55.7/0.041  3.5X 2X 1.58 1.09 P396L

In a specific embodiment, the invention encompasses an immunoglobulincomprising a variant Fc region with one or more amino acidmodifications, with an enhanced affinity for FcγRIIIA and/or FcγRIIAsuch that the immunoglobulin has an enhanced effector function, e.g.,antibody dependent cell mediated cytotoxicity, or phagocytosis. In aspecific embodiment, the one or more amino acid modifications whichincrease the affinity of the variant Fc region for FcγRIIIA and/orFcγRIIA and increase the ADCC activity of the immunoglobulin comprise asubstitution at position 379 with methionine; or a substitution atposition 243 with isoleucine and at position 379 with leucine; or asubstitution at position 288 with asparagine, at position 330 withserine, and at position 396 with leucine; or a substitution at position243 leucine and at position 255 with leucine; or a substitution atposition 334 with glutamic acid, at position 359 with asparagine, and atposition 366 with serine; or a substitution at position 288 withmethionine and at position 334 with glutamic acid; or a substitution atposition 334 with glutamic acid and at position 292 with leucine; or asubstitution at position 316 with aspartic acid, at position 378 withvaline, and at position 399 with glutamic acid; or a substitution atposition 315 with isoleucine, at position 379 with methionine, and atposition 399 with glutamic acid; or a substitution at position 243 withisoleucine, at position 379 with leucine, and at position 420 withvaline; or a substitution at position 247 with leucine and at position421 with lysine; or a substitution at position 248 with methionine; or asubstitution at position 392 with threonine and at position 396 withleucine; or a substitution at position 293 with valine, at position 295with glutamic acid, and at position 327 with threonine; or asubstitution at position 268 with asparagine and at position 396 withleucine; or a substitution at position 319 with phenylalanine, atposition 352 with leucine, and at position 396 with leucine; or asubstitution at position 243 with leucine, at position 292 with proline,at position 300 with leucine, at position 305 with isoleucine, and atposition 396 with leucine; or a substitution at position 243 withleucine, at position 292 with proline, at position 300 with leucine, andat position 396 with leucine; or a substitution at position 243 withleucine, at position 292 with proline, and at position 300 with leucine.

In another specific embodiment, the one or more amino acid modificationswhich increase the ADCC activity of the immunoglobulin is any of themutations listed below, in Table 8.

TABLE 8 AMINO ACID MODIFICATION WHICH INCREASE ADCC E333A, K334A T250S,P396L R292L, K334E P247S, P396L V379M K290E, V369A, T393A, P396L S219YK210N, K222I, K320M, P396L V282M L410H, P396L K222N Q419L, P396L F243I,V379L V427A, P396L F243L, R255L, E318K P217S, V305I, I309L, N390H, P396LK334I E258D, P396L K334E, T359N, T366S N384K, P396L K288M, K334E V323I,P396L K288N, A330S, P396L K246N, Q419R, P396L K326E V273I, K326E, L328I,P396L G316D, A378V, D399E K326I, S408N, P396L N315I, V379M, T394M K334N,P396L F243I, V379L, G420V V379M, P396L E293V, Q295E, A327T P227S, P396LY319F, P352L, P396L P217S, P396L K392T, P396L K261N, K210M, P396L K248MQ419H, P396L H268N, P396L K370E, P396L K290T, N390I, P396L L242F, P396LK326I, P396L F243L, V305I, A378D, F404S, P396L H268D, P396L R255L, P396LK210M, P396L V240A, P396L L358P, P396L P217A, T359A, P396L K288R, T307A,K344E, P396L P244H, P396L D270E, G316D, R416G V215I, K290V, P396L P247L,N421K F275L, Q362H, N384K, P396L P247L, N421K, D270E V305L, P396L Q419H,P396L, D270E S400F, P396L K370E, P396L, D270E V303I, P396L R255L, P396L,D270E F243L, R292P, Y300L, V305I, P396L V240A, P396L, D270E F243L,R292P, Y300L, P396L K392T, P396L, D270E F243L, R292P, Y300L

Alternatively or additionally, it may be useful to combine the aboveamino acid modifications or any other amino acid modifications disclosedherein with one or more further amino acid modifications that alter C1qbinding and/or complement dependent cytoxicity function of the Fcregion. The starting molecule of particular interest herein is usuallyone that binds to C1q and displays complement dependent cytotoxicity(CDC). The further amino acid substitutions described herein willgenerally serve to alter the ability of the starting molecule to bind toC1q and/or modify its complement dependent cytotoxicity function, e.g.,to reduce and preferably abolish these effector functions. However,molecules comprising substitutions at one or more of the describedpositions with improved C1q binding and/or complement dependentcytotoxicity (CDC) function are contemplated herein. For example, thestarting molecule may be unable to bind C1q and/or mediate CDC and maybe modified according to the teachings herein such that it acquiresthese further effector functions. Moreover, molecules with preexistingC1q binding activity, optionally further having the ability to mediateCDC may be modified such that one or both of these activities areenhanced.

As disclosed above, one can design an Fc region with altered effectorfunction, e.g., by modifying C1q binding and/or FcR binding and therebychanging CDC activity and/or ADCC activity. For example, one cangenerate a variant Fc region with improved C1q binding and improvedFcγRIII binding; e.g., having both improved ADCC activity and improvedCDC activity. Alternatively, where one desires that effector function bereduced or ablated, one may engineer a variant Fc region with reducedCDC activity and/or reduced ADCC activity. In other embodiments, one mayincrease only one of these activities, and optionally also reduce theother activity, e.g., to generate an Fc region variant with improvedADCC activity, but reduced CDC activity and vice versa.

The invention encompasses specific variants of the Fc region that havebeen identified using the methods of the invention from a yeast libraryof mutants after 2nd-4th-round of sorting are listed in Table 9. Table 9summarizes the various mutants that were identified using the methods ofthe invention. The mutants were assayed using an ELISA assay fordetermining binding to FcγRIIIA and FcγRIIB. The mutants were alsotested in an ADCC assay, by cloning the Fc variants into a ch 4-4-20antibody using methods disclosed and exemplified herein. Bolded itemsrefer to experiments, in which the ch4-4-20 were purified prior the ADCCassay. The antibody concentration used was in the range 0.5 μg/mL-1.0μg/mL.

TABLE 9 MUTATIONS IDENTIFIED IN THE Fc REGION 4-4-20 ADCC Binding toBinding to (Relative FcγRIIIA FcγRIIB Lysis Mutations Domain (ELISA)(ELISA) (Mut/Wt) pYD-CH1 library FACS screen with A tetramer Q347H;A339V CH3 ↑0.5x NT S415I; L251F CH2, CH3 ↑0.5x ↑.75x 0.82 K392R CH3 N/CNT D399E; R292L; V185M CH1, CH2, CH3 N/C ↑0.5x 0.65 0.9 K290E; L142PCH1, CH2 N/C NT R301C; M252L; S192T CH1, CH2 ↓.5x NT P291S; K288E;H268L: A141V CH1, CH2 ↓.5x NT N315I CH2 N/C ↑.75x S132I CH1 N/C NTS383N; N384K; T256N; V262L; K218E; R214I; All ↑0.5x NT K205E; F149Y;K133M S408I; V215I; V125L CH1, CH2, CH3 ↑0.5x ↑.75x 0.62 P396L CH3 ↑1x↑1x 0.55 G385E; P247H; CH2, CH3 ↑1x ↑.75x 0.44 P396H CH3 ↑1x ↑1x 0.58A162V CH1 N/C NT V348M; K334N; F275I; Y202M; K147T CH1, CH2, CH3 ↑0.5x↑.75x 0.33 H310Y; T289A; G337E CH2 ↑.5x NT S119F; G371S; Y407V; E258DCH1, CH2, CH3 N/C N/C 0.29 K409R; S166N CH1, CH3 N/C NT in vitro SiteDirected mutants R292L CH2 NT NT 0.82 T359N CH3 NT NT 1.06 T366S CH3 NTNT 0.93 E333A, K334A CH2 NT NT 1.41 R292L, K334E CH2 NT NT 1.41; 1.64R292L, P396L, T359N CH2, CH3 NT NT 0.89; 1.15 V379L CH3 NT NT 0.83 K288NCH2 NT NT 0.78 A330S CH2 NT NT 0.52 F243L CH2 NT NT 0.38 E318K CH2 NT NT0.86 K288N, A330S CH2 NT NT 0.08 R255L, E318K CH2 NT NT 0.82 F243L,E318K CH2 NT NT 0.07 Mutants in 4-4-20 mini-library Increased FcγRIIIAbinding, decreased or no change to FcγRIIB binding N/C means no change;N/B means no binding; NT means not tested V379M CH3 ↑2x N/C 1.47 S219YHinge ↑1x ↓ or N/B 1.28 V282M CH2 ↑1x ↓ or N/B 1.25; 1 F275I, K334N,V348M CH2 ↑0.5x N/C D401V CH3 ↑0.5x N/C V279L, P395S CH2 ↑1x N/C K222NHinge ↑1x ↓or N/B 1.33; 0.63 K246T, Y319F CH2 ↑1x N/C F243I, V379L CH2,CH3 ↑1.5x ↓ or N/B 1.86; 1.35 F243L, R255L, E318K CH2 ↑1x ↓ or N/B 1.81;1.45 K334I CH2 ↑1x N/C 2.1; 1.97 K334E, T359N, T366S CH2, CH3 ↑1.5x N/C1.49; 1.45 K288M, K334E CH2 ↑3x ↓ or N/B 1.61; 1.69 K334E, E380D CH2,CH3 ↑1.5x N/C T256S, V305I, K334E, N390S CH2, CH3 ↑1.5x N/C K334E CH2↑2.5x N/C 1.75; 2.18 T335N, K370E, A378V, T394M, S424L CH2, CH3 ↑0.5xN/C E233D, K334E CH2 ↑1.5x N/C 0.94; 1.02 K334E, T359N, T366S, Q386R CH2↑1x N/C Increased Binding to FcγIIIA and FcγRIIB K246T, P396H CH2, CH3↑1x ↑2.5x H268D, E318D CH2 ↑1.5x ↑5x K288N, A330S, P396L CH2, CH3 ↑5x↑3x 2.34; 1.66; 2.54 I377F CH3 ↑1.5x ↑0.5x P244H, L358M, V379M, N384K,V397M CH2, CH3 ↑1.75x ↑1.5x P217S, A378V, S408R Hinge, CH3 ↑2x ↑4.5xP247L, I253N, K334N CH2 ↑3x ↑2.5x P247L CH2 ↑0.5x ↑4x 0.91; 0.84 F372YCH3 ↑0.75x ↑5.5x 0.88; 0.59 K326E CH2 ↑2x ↑3.5x 1.63; 2 K246I, K334N CH2↑0.5x ↑4x 0.66; 0.6 K320E, K326E CH2 ↑1x ↑1x H224L Hinge ↑0.5x ↑5x 0.55;0.53 S375C, P396L CH3 ↑1.5x ↑4.5x D312E, K327N, I378S CH2, CH3 ↑0.5x N/CK288N, K326N CH2 ↑1x N/C F275Y CH2 ↑3x N/C 0.64 P247L, N421K CH2, CH3↑3x N/C 2.0 S298N, W381R CH2, CH3 ↑2x N/C D280E, S354F, A431D, L441ICH2, CH3 ↑3x N/C 0.62 R255Q, K326E CH2 ↑2x N/C 0.79 K218R, G281D, G385RH, CH2, CH3 ↑3.5x N/C 0.67 L398V CH3 ↑1.5x N/C P247L, A330T, S440G CH2,CH3 ↑0.75x ↓ 0.25x V284A, F372L CH2, CH3 1x N/C T335N, P387S, H435Q CH2,CH3 1.25x N/C P247L, A431V, S442F CH2, CH3 1x N/C Increased Binding toFcγRIIIA and FcγRIIB P343S, P353L, S375I, S383N CH3 ↑0.5x ↑6x T394M,V397M CH3 ↑0.5x ↑3x E216D, E345K, S375I H, CH2, CH3 ↑0.5x ↑4x K334N, CH2↑0.5x ↑2x K288N, A330S, P396L CH2, CH3 ↑0.5x ↑9x P247L, E389G CH2, CH3↑1.5x ↑9x K222N, T335N, K370E, A378V, T394M H, CH2, CH3 ↑1x ↑7x G316D,A378V, D399E CH2, CH3 ↑1.5x ↑14x 2.24 N315I, V379M, T394M CH2, CH3 ↑1x↑9x 1.37 K290T, G371D, CH2, CH3 ↑0.25x ↑6x P247L, L398Q CH2, CH3 ↑1.25x↑10x K326Q, K334E, T359N, T366S CH2, CH3 ↑1.5x ↑5x S400P CH3 ↑1x ↑6xP247L, I377F CH2, CH3 ↑1x ↑5x A378V, N390I, V422I CH3 ↑0.5x ↑5x K326E,G385E CH2, CH3 ↑0.5x ↑15x V282E, V369I, L406F CH2, CH3 ↑0.5x ↑7x V397M,T411A, S415N CH3 ↑0.25x ↑5x T223I, T256S, L406F H, CH2, CH3 ↑0.25x ↑6xS298N, S407R CH2, CH3 ↑0.5x ↑7x K246R, S298N, I377F CH2, CH3 ↑1x ↑5xS407I CH3 ↑0.5x ↑4x F372Y CH3 ↑0.5x ↑4x L235P, V382M, S304G, V305I,V323I CH2, CH3 ↑2x ↑2x P247L, W313R, E388G CH2, CH3 ↑1.5x ↑1x D221Y,M252I, A330G, A339T, T359N, V422I, H433L H, CH2, CH3 ↑2.5x ↑6x E258D,N384K CH2, CH3 ↑1.25x ↑4x F241L, E258G CH2 ↑2x ↑2.5x −0.08 K370N, S440NCH3 ↑1x ↑3.5x K317N, F423-deleted CH2, CH3 ↑2.5x ↑7x 0.18 F243I, V379L,G420V CH2, CH3 ↑2.5x ↑3.5x 1.35 P227S, K290E H, CH2 ↑1x ↑0.5x A231V,Q386H, V412M CH2, CH3 ↑1.5x ↑6x T215P, K274N, A287G, K334N, L365V, P396LH, CH2, CH3 ↑2x ↑4x Increased Binding to FcγRIIB but not FcγRIIIA K334E,E380D CH2, CH3 N/C ↑4.5x T366N CH3 N/C ↑5x P244A, K326I, C367R, S375I,K447T CH2, CH3 N/C ↑3x C229Y, A287T, V379M, P396L, L443V H, CH2, CH3 ↓0.25x ↑10x Decreased binding to FcγRIIIA and FcγRIIB R301H, K340E, D399ECH2, CH3 ↓ 0.50x ↓ 0.25x K414N CH3 ↓ 0.25x N/B P291S, P353Q CH2, CH3 ↓0.50x ↓ 0.25x V240I, V281M CH2 ↓ 0.25x ↓ 0.25x P232S, S304G CH2 N/B N/BE269K, K290N, Q311R, H433Y CH2, CH3 N/B N/B M352L CH3 N/B N/B E216D,K334R, S375I H, CH2, CH3 N/B N/B P247L, L406F CH2, CH3 N/B N/B T335N,P387S, H435Q CH2, CH3 N/B N/B T225S CH2 ↓ 0.25x ↓ 0.50x D399E, M428L CH3↓ 0.50x ↓ 0.50x K246I, Q362H, K370E CH2, CH3 N/B ↓ 0.50x K334E, E380D,G446V CH2, CH3 N/B N/B I377N CH3 ↓ 0.50x N/B V303I, V369F, M428L CH2,CH3 N/B N/B L251F, F372L CH2, CH3 N/B N/B K246E, V284M, V308A CH2, CH3N/B N/B D399E, G402D CH3 N/B N/B D399E, M428L CH3 N/B N/B FcγRIIBdepletion/FcγRIIIA selection: Naive Fc library. E293V, Q295E, A327T CH2↑0.4x ↓ or N/B 4.29 Y319F, P352L, P396L CH2, CH3 ↑3.4x ↑2x 1.09 K392T,P396L CH3 ↑4.5x ↑2.5x 3.07 K248M CH2 ↑0.4x ↓ or N/B 4.03 H268N, P396LCH2, CH3 ↑2.2x ↑4.5x 2.24 Solution competition 40X FcγRIIB-G2: P396LLibrary D221E, D270E, V308A, Q311H, P396L, G402D ↑3.6x ↑0.1x 3.17Equilibrium Screen: 0.8 μM FcγRIIIA monomer: P396L library K290T, N390I,P396L CH2, CH3 ↑2.8x ↑6.1x 1.93 K326I, P396L CH2, CH3 ↑2.9x ↑5.9x 1.16H268D, P396L CH2, CH3 ↑3.8x ↑13.7x 2.15 K210M, P396L CH1, CH3 ↑1.9x↑4.6x 2.02 L358P, P396L CH3 ↑1.9x ↑4.2x 1.58 K288R, T307A, K344E, P396LCH2, CH3 ↑4.1x ↑2.3x 3.3 V273I, K326E, L328I, P396L CH2, CH3 ↑1.3x↑10.8x 0.78 K326I, S408N, P396L CH2, CH3 ↑4x ↑9.3x 1.65 K334N, P396LCH2, CH3 ↑3.1x ↑3x 2.43 V379M, P396L CH3 ↑1.9x ↑5.6x 2.01 P227S, P396LCH2, CH3 ↑1.5x ↑4x 2.01 P217S, P396L H, CH3 ↑1.6x ↑4.5x 2.04 K261N,K210M, P396L CH2, CH3 ↑2x ↑4.2x 2.06 Kinetic Screen: O.8 μM, 1′ withcold 8 μM FcγRIIIA: P396L Library term is M, P396L CH3 ↑1.9x ↑7.2x 3.09Q419H, P396L CH3 ↑2x ↑6.9x 2.24 K370E, P396L CH3 ↑2x ↑6.6x 2.47 L242F,P396L CH2, CH3 ↑2.5x ↑4.1x 2.4 F243L, V305I, A378D, F404S, P396L CH2,CH3 ↑1.6x ↑5.4x 3.59 R255L, P396L CH2, CH3 ↑1.8x ↑6x 2.79 V240A, P396LCH2, CH3 ↑1.3x ↑4.2x 2.35 T250S, P396L CH2, CH3 ↑1.5x ↑6.8x 1.60 P247S,P396L CH2, CH3 ↑1.2x ↑4.2x 2.10 K290E, V369A, T393A, P396L CH2, CH3↑1.3x ↑6.7x 1.55 K210N, K222I, K320M, P396L H, CH2, CH3 ↑2.7x ↑8.7x 1.88L410H, P396L CH3 ↑1.7x ↑4.5x 2.00 Q419L, P396L CH3 ↑2.2x ↑6.1x 1.70V427A, P396L CH3 ↑1.9x ↑4.7x 1.67 P217S, V305I, I309L, N390H, P396L H,CH2, CH3 ↑2x ↑7x 1.54 E258D, P396L CH2, CH3 ↑1.9x ↑4.9x 1.54 N384K,P396L CH3 ↑2.2x ↑5.2x 1.49 V323I, P396L CH2, CH3 ↑1.1x ↑8.2x 1.29 K246N,Q419R, P396L CH2, CH3 ↑1.1x ↑4.8x 1.10 P217A, T359A, P396L H, CH2, CH3↑1.5x ↑4.8x 1.17 P244H, P396L CH2, CH3 ↑2.5x ↑4x 1.40 V215I, K290V,P396L H, CH2, CH3 ↑2.2x ↑4.6x 1.74 F275L, Q362H, N384K, P396L CH2, CH3↑2.2x ↑3.7x 1.51 V305L, P396L CH2, CH3 ↑1.3x ↑5.5x 1.50 S400F, P396L CH3↑1.5x ↑4.7x 1.19 V303I, P396L CH3 ↑1.1x ↑4x 1.01 FcγRIIB depletionFcγRIIIA 158V solid phase selection: Naïve Library A330V, H433Q, V427MCH2, CH3 NT NT NT V263Q, E272D, Q419H CH2, CH3 NT NT NT N276Y, T393N,W417R CH2, CH3 NT NT NT V282L, A330V, H433Y, T436R CH2, CH3 NT NT NTA330V, Q419H CH2, CH3 NT NT NT V284M, S298N, K334E, R355W CH2, CH3 NT NTNT A330V, G427M, K438R CH2, CH3 NT NT NT S219T, T225K, D270E, K360R CH2,CH3 NT NT NT K222E, V263Q, S298N CH2 NT NT NT V263Q, E272D CH2 NT NT NTR292G CH2 NT NT NT S298N CH2 NT NT NT E233G, P247S, L306P CH2 NT NT NTD270E CH2 NT NT NT S219T, T225K, D270E CH2 NT NT NT K326E, A330T CH2 NTNT NT E233G CH2 NT NT NT S254T, A330V, N361D, P243L CH2, CH3 NT NT NTFcγRIIB depletion FcγRIIIA 158F solid phase selection: Naïve Library158F by FACS top 0.2% V284M, S298N, K334E, R355W R416T CH2, CH3 NT NTFcγRIIB depletion FcgRIIA 131H solid phase selection: Naïve LibraryR292P, V305I CH2, CH2 NT NT D270E, G316D, R416G CH2, CH3 NT NT V284M,R292L, K370N CH2, CH3 NT NT R292P, V305I, F243L CH2 NT NT

In preferred embodiments, the invention provides modified immunoglobulinmolecules (e.g., antibodies) with variant Fc regions, having one or moreamino acid modifications, which one or more amino acid modificationsincrease the affinity of the molecule for FcγRIIIA and/or FcγRIIA. Suchimmunoglobulins include IgG molecules that naturally contain FcγRbinding regions (e.g., FcγRIIIA and/or FcγRIIB binding region), orimmunoglobulin derivatives that have been engineered to contain an FcγRbinding region (e.g., FcγRIIIA and/or FcγRIIB binding region). Themodified immunoglobulins of the invention include any immunoglobulinmolecule that binds, preferably, immunospecifically, i.e., competes offnon-specific binding as determined by immunoassays well known in the artfor assaying specific antigen-antibody binding, an antigen and containsan FcγR binding region (e.g., a FcγRIIIA and/or FcγRIIB binding region).Such antibodies include, but are not limited to, polyclonal, monoclonal,bi-specific, multi-specific, human, humanized, chimeric antibodies,single chain antibodies, Fab fragments, F(ab′)₂ fragments,disulfide-linked Fvs, and fragments containing either a VL or VH domainor even a complementary determining region (CDR) that specifically bindsan antigen, in certain cases, engineered to contain or fused to an FcγRbinding region.

In some embodiments, the molecules of the invention comprise portions ofan Fc region. As used herein the term “portion of an Fc region” refersto fragments of the Fc region, preferably a portion with effectoractivity and/or FcγR binding activity (or a comparable region of amutant lacking such activity). The fragment of an Fc region may range insize from 5 amino acids to the entire Fc region minus one amino acid.The portion of an Fc region may be missing up to 10, up to 20, up to 30amino acids from the N-terminus or C-terminus.

The IgG molecules of the invention are preferably IgG1 subclass of IgGs,but may also be any other IgG subclasses of given animals. For example,in humans, the IgG class includes IgG1, IgG2, IgG3, and IgG4; and mouseIgG includes IgG1, IgG2a, IgG2b, IgG2c and IgG3.

The immunoglobulins (and other polypeptides used herein) may be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulin and that do not express endogenous immunoglobulins, asdescribed infra and, for example, in U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide or may be specificfor heterologous epitopes, such as a heterologous polypeptide or solidsupport material. See, e.g., PCT publications WO 93/17715; WO 92/08802;WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol., 147:60-69, 1991;U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al., J. Immunol., 148:1547-1553, 1992.

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by theinstant invention. Examples of BsAbs include without limitation thosewith one arm directed against a tumor cell antigen and the other armdirected against a cytotoxic molecule.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983); which is incorporated herein by reference inits entirety). Because of the random assortment of immunoglobulin heavyand light chains, these hybridomas (quadromas) produce a potentialmixture of 10 different antibody molecules, of which only one has thecorrect bispecific structure. Purification of the correct molecule,which is usually done by affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in WO 93/08829, and in Traunecker et al., EMBO J.,10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when, the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986). According to anotherapproach described in WO96/27011, a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant cell culture. The preferred interfacecomprises at least a part of the CH3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. See, e.g., Tutt et al. J.Immunol. 147: 60 (1991), which is incorporated herein by reference.

The antibodies of the invention include derivatives that are otherwisemodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom binding antigen and/or generating an anti-idiotypic response. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al.,BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods,125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions andconstant domains from a human immunoglobulin molecule. Often, frameworkresidues in the human framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. See, e.g., Queen etal., U.S. Pat. No. 5,585,089; Riechmann et al., Nature, 332:323, 1988,which are incorporated herein by reference in their entireties.Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology,28(4/5):489-498, 1991; Studnicka et al., Protein Engineering,7(6):805-814, 1994; Roguska et al., Proc Natl. Acad. Sci. USA,91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332), all ofwhich are hereby incorporated by reference in their entireties.Humanized antibodies may be generated using any of the methods disclosedin U.S. Pat. No. 5,693,762 (Protein Design Labs), U.S. Pat. No.5,693,761, (Protein Design Labs) U.S. Pat. No. 5,585,089 (Protein DesignLabs), U.S. Pat. No. 6,180,370 (Protein Design Labs), and U.S.Publication Nos. 20040049014, 200300229208, each of which isincorporated herein by reference in its entirety.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol., 13:65-93, 1995. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., Bio/technology,12:899-903, 1988).

The invention encompasses engineering human or humanized therapeuticantibodies (e.g., tumor specific monoclonal antibodies) in the Fcregion, by modification (e.g., substitution, insertion, deletion) of atleast one amino acid residue, which modification increases the affinityof the Fc region for FcγRIIIA and/or FcγRIIA. In another embodiment, theinvention relates to engineering human or humanized therapeuticantibodies (e.g., tumor specific monoclonal antibodies) in the Fcregion, by modification of at least one amino acid residue, whichmodification increases the affinity of the Fc region for FcγRIIIA and/orFcγRIIA and further decreases the affinity of the Fc region for FcγRIIB.The engineered therapeutic antibodies may further have an enhancedeffector function, e.g., enhanced ADCC activity, phagocytosis activity,etc., as determined by standard assays known to those skilled in theart.

In a specific embodiment, the invention encompasses engineering ahumanized monoclonal antibody specific for Her2/neu protooncogene (e.g.,Ab4D5 humanized antibody as disclosed in Carter et al., 1992, Proc.Natl. Acad. Sci. USA 89:4285-9) by modification (e.g., substitution,insertion, deletion) of at least one amino acid residue whichmodification increases the affinity of the Fc region for FcγRIIIA and/orFcγRIIA. In another specific embodiment, modification of the humanizedHer2/neu monoclonal antibody may also further decrease the affinity ofthe Fc region for FcγRIIB. In yet another specific embodiment, theengineered humanized monoclonal antibodies specific for Her2/neu mayfurther have an enhanced effector function as determined by standardassays known in the art and disclosed and exemplified herein.

In another specific embodiment, the invention encompasses engineering amouse human chimeric anti-CD20 monoclonal antibody, 2H7 by modification(e.g., substitution, insertion, deletion) of at least one amino acidresidue which modification increases the affinity of the Fc region forFcγRIIIA and/or FcγRIIA. In another specific embodiment, modification ofthe anti-CD20 monoclonal antibody, 2H7 may also further decrease theaffinity of the Fc region for FcγRIIB. In yet another specificembodiment, the engineered anti-CD20 monoclonal antibody, 2H7 mayfurther have an enhanced effector function as determined by standardassays known in the art and disclosed and exemplified herein.

In another specific embodiment, the invention encompasses engineering anantibody that binds A33, CD5, CD11c, CD19, CD20, CD22, CD23, CD27, CD40,CD45, CD79a, CD79b, CD103, CTLA4, ErbB1, ErbB3, ErbB4, VEGF receptor,TNF-a receptor, TNF-β receptor, or INF-γ receptor (particularly ahumanized or chimerized form of the antibody) by modification (e.g.,substitution, insertion, deletion) of at least one amino acid residuewhich modification increases the affinity of the Fc region for FcγRIIIAand/or FcγRIIA. In another specific embodiment, modification of theantibody that binds A33, CD5, CD11c, CD19, CD20, CD22, CD23, CD27, CD40,CD45, CD79a, CD79b, CD103, CTLA4, ErbB1, ErbB3, ErbB4, VEGF receptor,TNF-a receptor, TNF-β receptor, or INF-γ receptor may also furtherdecrease the affinity of the Fc region for FcγRIIB. In yet anotherspecific embodiment, the antibody that binds A33, CD5, CD11c, CD19,CD20, CD22, CD23, CD27, CD40, CD45, CD79a, CD79b, CD103, CTLA4, ErbB1,ErbB3, ErbB4, VEGF receptor, TNF-a receptor, INF-β receptor, or INF-γreceptor may further have an enhanced effector function as determined bystandard assays known in the art and disclosed and exemplified herein.

In certain embodiments, the invention encompasses engineering anantibody (or chimeric, humanized or other engineered versions thereof),comprising the heavy chain variable domain and/or light chain variabledomain of the monoclonal antibody produced by clone 2B6, 3H7, 8B5.4.3,1D5, 2E1, 2H9, 2D11 or 1F2 having ATCC accession numbers PTA-4591,PTA-4592, PTA-7610, PTA-5958, PTA-5961, PTA-5962, PTA-5960, andPTA-5959, respectively (deposited at ATCC, 10801 University Boulevard,Manassas, Va. 02209-2011, all of which are incorporated herein byreference). In a specific embodiment, the invention encompassesengineering a humanized antibody comprising the heavy chain variableand/or light chain variable domains of 2B6, 3H7 or 8B5.3.4. In anotherspecific embodiment, the invention encompasses engineering a humanizedantibody comprising the CDRs of 2B6, 3H7 or 8B5.3.4. In another specificembodiment, the invention encompasses engineering a humanized antibodycomprising the heavy chain variable domain having the amino acidsequence of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3 and the light chainvariable domain having the amino acid sequence of SEQ ID NO: 4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8. In another specificembodiment, the invention encompasses engineering an anti-FcγRIIBantibody comprising the heavy chain variable domain having the aminoacid sequence of SEQ ID NO:13 and the light chain variable domain havingthe amino acid sequence of SEQ ID NO:14. In another specific embodiment,the invention encompasses engineering a humanized anti-FcγRIIB antibodycomprising the heavy chain variable domain having the amino acidsequence of SEQ ID NO:3 and the light chain variable domain having theamino acid sequence of SEQ ID NO:8. In another specific embodiment, theinvention encompasses engineering a humanized anti-FcγRIIB antibodycomprising the heavy chain variable domain having the amino acidsequence of SEQ ID NO:9 and the light chain variable domain having theamino acid sequence of SEQ ID NO:10.

In another specific embodiment, the invention encompasses engineering ananti-FcγRIIB antibody including but not limited to any of the antibodiesdisclosed in U.S. Provisional Application No. 60/403,266 filed on Aug.12, 2002, U.S. application Ser. No. 10/643,857 filed on Aug. 14, 2003,U.S. Provisional Application No. 60/562,804 filed on Apr. 16, 2004, U.S.Provisional Application No. 60/582,044 filed on Jun. 21, 2004, U.S.Provisional Application No. 60/582,045 filed on Jun. 21, 2004, U.S.Provisional Application No. 60/636,663 filed on Dec. 15, 2004 and U.S.application Ser. No. 10/524,134 filed Feb. 11, 2005 by modification(e.g., substitution, insertion, deletion) of at least one amino acidresidue which modification increases the affinity of the Fc region forFcγRIIIA and/or FcγRIIA. In another specific embodiment, the inventionencompasses engineering a humanized anti-FcγRIIB antibody including butnot limited to any of the antibodies disclosed in U.S. ProvisionalApplication No. 60/569,882 filed on May 10, 2004, U.S. ProvisionalApplication No. 60/582,043 filed on Jun. 21, 2004 and U.S. applicationSer. No. 11/126,978, filed on May 10, 2005 by modification (e.g.,substitution, insertion, deletion) of at least one amino acid residuewhich modification increases the affinity of the Fc region for FcγRIIIAand/or FcγRIIA. Each of the above mentioned applications is incorporatedherein by reference in its entirety. Examples of anti-FcγRIIBantibodies, which may or may not be humanized, that may be engineered inaccordance with the methods of the invention are 2B6 monoclonal antibodyhaving ATCC accession number PTA-4591 and 3H7 having ATCC accessionnumber PTA-4592, 1D5 monoclonal antibody having ATCC accession numberPTA-5958, 1F2 monoclonal antibody having ATCC accession number PTA-5959,2D11 monoclonal antibody having ATCC accession number PTA-5960, 2E1monoclonal antibody having ATCC accession number PTA-5961, 8B5.3.4having ATCC accession number PTA-7610, and 2H9 monoclonal antibodyhaving ATCC accession number PTA-5962 (all deposited at 10801 UniversityBoulevard, Manassas, Va. 02209-2011), which are incorporated herein byreference. In another specific embodiment, modification of theanti-FcγRIIB antibody may also further decrease the affinity of the Fcregion for FcγRIIB. In yet another specific embodiment, the engineeredanti-FcγRIIB antibody may further have an enhanced effector function asdetermined by standard assays known in the art and disclosed andexemplified herein.

In a specific embodiment, the invention encompasses engineering ananti-FcγRIIB antibody according to methods of the present invention thatcomprises one or more complementarily determining regions (CDRs),preferably all 6 CDRs, of the antibody produced by clone 2B6, 3H7, or8B5.3.4 with ATCC accession numbers PTA-4591, PTA-4592, and PTA-7610,respectively (e.g., the heavy chain CDR3). In a specific embodiment, ananti-FcγRIIB antibody engineered according to methods of the inventioncomprises one or more complementarily determining regions (CDRs),preferably all 6 CDRs, of the antibody produced by clone 1D5, 2E1, 2H9,2D11, and 1F2 having ATCC Accession numbers, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively (e.g., the heavy chainCDR3). In another embodiment, an anti-FcγRIIB antibody engineeredaccording to methods of the invention binds to the same epitope as themouse monoclonal antibody produced from clone 2B6, 3H7, or 8B5.3.4 withATCC accession numbers PTA-4591, PTA-4592, and PTA-7610, respectivelyand/or competes with the mouse monoclonal antibody produced from clone2B6, 3H7, or 8B5.3.4 with ATCC accession numbers PTA-4591, PTA-4592, andPTA-7610, respectively as determined, e.g., in an ELISA assay or otherappropriate competitive immunoassay, and also binds FcγRIIB with agreater affinity than said antibody or a fragment thereof binds FcγRIIA.In another embodiment, an anti-FcγRIIB antibody engineered according tomethods of the invention binds to the same epitope as the mousemonoclonal antibody produced from clone 1D5, 2E1, 2H9, 2D11, and 1F2having ATCC Accession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960,and PTA-5959, respectively, and/or competes with the mouse monoclonalantibody produced from clone 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCCAccession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively, as determined, e.g., in an ELISA assay or otherappropriate competitive immunoassay, and also binds FcγRIIB, via itsvariable region, with a greater affinity than said antibody or afragment thereof binds FcγRIIA.

The present invention also encompasses engineering an anti-FcγRIIBantibody comprising a heavy chain variable domain and/or light chainvariable domain amino acid sequence that is at least 45%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of the heavy chain variable domainand/or light chain variable domain of the mouse monoclonal antibodyproduced by clone 2B6, 3H7, 8B5.3.4, 1D5, 2E1, 2H9, 2D11, or 1F2 havingATCC accession numbers PTA-4591, PTA-4592, PTA-7610, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively. The present inventionfurther encompasses the engineering of anti-FcγRIIB antibodiescomprising an amino acid sequence of one or more CDRs that is at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99% identical to the amino acid sequence of one or moreCDRs of the mouse monoclonal antibody produced by clone 2B6, 3H7,8B5.3.4, 1D5, 2E1, 2H9, 2D11, or 1F2 having ATCC accession numbersPTA-4591, PTA-4592, PTA-7610, PTA-5958, PTA-5961, PTA-5962, PTA-5960,and PTA-5959, respectively. The determination of percent identity of twoamino acid sequences can be determined by any method known to oneskilled in the art, including BLAST protein searches.

The present invention also encompasses the engineering of one or moreanti-FcγRIIB antibodies comprising one or more variable domains encodedby a nucleotide sequence that hybridizes to the nucleotide sequence ofone or more variable domains of a mouse monoclonal antibody produced byclone 2B6, 3H7, 8B5.3.4, 1D5, 2E1, 2H9, 2D11, or 1F2 having ATCCaccession numbers PTA-4591, PTA-4592, PTA-7610, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively, under stringentconditions. In a preferred embodiment, the invention encompassesengineering one or more anti-FcγRIIB antibodies comprising a variablelight chain and/or variable heavy chain domain encoded by a nucleotidesequence that hybridizes under stringent conditions to the nucleotidesequence of the variable light chain and/or variable heavy chain domainof the mouse monoclonal antibody produced by clone 2B6, 3H7, 8B5.3.4,1D5, 2E1, 2H9, 2D11, or 1F2 having ATCC accession numbers PTA-4591,PTA-4592, PTA-7610, PTA-5958, PTA-5961, PTA-5962, PTA-5960, andPTA-5959, respectively, under stringent conditions. In another preferredembodiment, the invention provides engineering anti-FcγRIIB antibodiescomprising one or more CDRs encoded by a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequence of oneor more CDRs of the mouse monoclonal antibody produced by clone 2B6,3H7, 8B5.3.4, 1D5, 2E1, 2H9, 2D11, or 1F2 having ATCC accession numbersPTA-4591, PTA-4592, PTA-7610, PTA-5958, PTA-5961, PTA-5962, PTA-5960,and PTA-5959, respectively. Stringent hybridization conditions include,but are not limited to, hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C., highly stringentconditions such as hybridization to filter-bound DNA in 6×SSC at about45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 60°C., or any other stringent hybridization conditions known to thoseskilled in the art (see, for example, Ausubel, F. M. et al., eds. 1989Current Protocols in Molecular Biology, vol. 1, Green PublishingAssociates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to6.3.6 and 2.10.3, incorporated herein by reference).

In a preferred embodiment, the engineered antibodies of the inventionare humanized by any method known in the art or described herein and/orcomprise the CDR regions of a humanized FcγRIIB specific antibody orhumanized CD20 specific antibody, wherein said CDRs are derived from amurine antibody specific for FcγRIIB or CD20, respectively. In someembodiments, the humanized antibodies described herein comprisealterations, including but not limited to amino acid deletions,insertions, modifications, of the acceptor antibody, i.e., human, heavyand/or light chain variable domain framework regions that are necessaryfor retaining binding specificity of the donor monoclonal antibody. Insome embodiments, the framework regions of the humanized antibodiesdescribed herein do not necessarily consist of the precise amino acidsequence of the framework region of a natural occurring human antibodyvariable region, but contains various alterations, including but notlimited to amino acid deletions, insertions, modifications that alterthe property of the humanized antibody, for example, improve the bindingproperties of a humanized antibody variable region specific for the sametarget as the murine FcγRIIB or CD20 specific antibody. In mostpreferred embodiments, a minimal number of alterations are made to theframework region in order to avoid large-scale introductions ofnon-human framework residues and to ensure minimal immunogenicity of thehumanized antibody of the invention in humans. The donor monoclonalantibody is preferably a monoclonal antibody produced by clones 2B6,3H7, 8B5.3.4, 1D5, 2E1, 2H9, 2D11, or 1F2 (having ATCC accession numbersPTA-4591, PTA-4592, PTA-7610, PTA-5958, PTA-5961, PTA-5962, PTA-5960,and PTA-5959, respectively) which bind FcγRIIB, or the monoclonalantibody is a CD20 antibody, such as rituximab or 2H7.

In a specific embodiment, the invention encompasses engineering aCDR-grafted antibody that comprises a heavy chain variable region domaincomprising framework residues of the recipient antibody and residuesfrom the donor monoclonal antibody, which specifically binds FcγRIIB,e.g., monoclonal antibody produced from clones 2B6, 3H7, 8B5.3.4, 1D5,2E1, 2H9, 2D11, or 1F2 having ATCC accession numbers PTA-4591, PTA-4592,PTA-7610, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively. In another specific embodiment, the invention encompassesengineering a CDR-grafted antibody that comprises a light chain variableregion domain comprising framework residues of the recipient antibodyand residues from the donor monoclonal antibody, which specificallybinds FcγRIIB, e.g., monoclonal antibody produced from clones 2B6, 3H7,8B5.3.4, 1D5, 2E1, 2H9, 2D11, or 1F2.

Preferably the FcγRIIB humanized antibodies bind the extracellulardomain of native human FcγRIIB. The humanized anti-FcγRIIB antibodies ofthe combinations can have a heavy chain variable region comprising theamino acid sequence of CDR1 (SEQ ID NO: 15 or SEQ ID NO: 16) and/or CDR2(SEQ ID NO:17 or SEQ ID NO:18) and/or CDR3 (SEQ ID NO: 19 or SEQ IDNO:20) and/or a light chain variable region comprising the amino acidsequence of CDR1 (SEQ ID NO:21 or SEQ ID NO:22) and/or a CDR2 (SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26) and/or CDR3 (SEQ IDNO:27 or SEQ ID NO:28).

In a specific embodiment, the invention encompasses the engineering of ahumanized anti-FcγRIIB antibody with the heavy chain variable domainhaving the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3 and a light chain variable domain having the amino acid sequence ofSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7 or SEQ ID NO:8.

In one specific embodiment, the invention encompasses engineering ahumanized anti-FcγRIIB antibody, wherein the VH region of the FcγRIIBantibody consists of the FR segments from the human germline VH segmentVH1-18 (Matsuda et al., 1998, J. Exp. Med. 188:2151062) and JH6 (Ravetchet al., 1981, Cell 27(3 Pt. 2): 583-91), and one or more CDR regions ofa 2B6 VH, having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:17,or SEQ ID NO:19. In one embodiment, the 2B6 VH has the amino acidsequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:29. In anotherspecific embodiment, the humanized anti-FcγRIIB antibody furthercomprises a VL region, which consists of the FR segments of the humangermline VL segment VK-A26 (Lautner-Rieske et al., 1992, Eur. J.Immunol. 22:1023-1029) and JK4 (Hieter et al., 1982, J. Biol. Chem.257:1516-22), and one or more CDR regions of a 2B6VL, having the aminoacid sequence of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,and SEQ ID NO:27. In one embodiment, the 2B6 VL has the amino acidsequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7, SEQ IDNO:8, or SEQ ID NO:30, and optionally in combination with one of theabove-referenced 2B6 VH.

In some embodiments, the anti-FcγRIIB antibody engineered in accordancewith the methods of the invention has a VH chain and/or a VH domaincomprising the amino acid sequence (H2B6VH-3) (SEQ ID NO:3):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWIGVIDPSDTYPNYNKKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCARNG DSDYYSGMDYWGQGTTVTVSS

In some embodiments, the anti-FcγRIIB antibody engineered in accordancewith the methods of the invention has a VL chain and/or VL domaincomprising the amino acid sequence (H2B6VL-5) (SEQ ID NO:8):

EIVLTQSPDFQSVTPKEKVTFTCRTSQSIGTNIHWYQQKPDQSPKLLIKEVSESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNTWPFTFGG GTKVEIK

In some embodiments, the anti-FcγRIIB antibody engineered in accordancewith the methods of the invention has a VH chain and/or VH domaincomprising the amino acid sequence (H2B6VH-3):

(SEQ ID NO: 3) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWIGVIDPSDTYPNYNKKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCARNGDSDYYSGMDYWGQGTTVTVSS,and a VL chain and/or VL domain comprising the amino acid sequence(H2B6VL-5) (SEQ ID NO:8):

EIVLTQSPDFQSVTPKEKVTFTCRTSQSIGTNIHWYQQKPDQSPKLLIKEVSESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNTWPFTFGG GTKVEIK

In some embodiments, the anti-FcγRIIB antibody engineered in accordancewith the methods of the invention has a VH domain and/or VH chaincomprising the amino acid sequence (8B5.3.4 VH, see FIG. 2):

(SEQ ID NO: 9) EVKLEESGGGLVQPGGSMKLSCEASGFTFSDAWMDWVRQSPEKGLEWVAEIRNKAKNHATYYAESVIGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCGA LGLDYWGQGTTLTVSS.

In some embodiments, the anti-FcγRIIB antibody engineered in accordancewith the methods of the invention has a VL domain and/or VL chaincomprising the amino acid sequence (8B5.3.4 VL, see FIG. 3) (SEQ IDNO:10):

DIQMTQSPSSLLAALGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSESGSDYSLTISSLESEDFADYYCLQYFSYPLTFGA GTKLELK

In some embodiments, the anti-FcγRIIB antibody engineered in accordancewith the methods of the invention has a VH domain and/or VH chaincomprising the amino acid sequence (8B5.3.4 VH) (SEQ ID NO:9, see FIG.2):

EVKLEESGGGLVQPGGSMKLSCEASGFTFSDAWMDWVRQSPEKGLEWVAEIRNKAKNHATYYAESVIGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCGA LGLDYWGQGTTLTVSS,and a VL domain and/or VL chain comprising the amino acid sequence(8B5.3.4 VL, see FIG. 3) (SEQ ID NO:10):

DIQMTQSPSSLLAALGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSESGSDYSLTISSLESEDFADYYCLQYFSYPLTFGA GTKLELK

In another specific embodiment, the anti-FcγRIIB antibody engineered inaccordance with the methods of the invention is a humanized 3H7antibody, wherein the FcγRIIB VH region consists of the FR segments froma human germline VH segment and the CDR regions of the 3H7 VH, havingthe amino acid sequence of SEQ ID NO: 37. In another specificembodiment, the humanized 3H7 antibody further comprises a VL region,which consists of the FR segments of a human germline VL segment and theCDR regions of 3H7VL, having the amino acid sequence of SEQ ID NO:7.

In particular, the invention encompasses the engineering of ananti-FcγRIIB antibody wherein the antibody immunospecifically binds toan extracellular domain of native human FcγRIIB, said FcγRIIB antibodycomprising (or alternatively, consisting of) CDR sequences of 2B6, 3H7,or 8B5.3.4 in any of the following combinations: a VH CDR1 and a VLCDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VH CDR2 and aVL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and aVH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, aVH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, aVH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, aVH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; a VH CDR1, aVL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, aVL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, aVL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, aVH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and aVL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VHCDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VHCDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VLCDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VHCDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VLCDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VHCDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combination thereof ofthe VH CDRs and VL CDRs disclosed herein.

In a specific embodiment, the anti-FcγRIIB monoclonal antibody comprisesa modification at position 334 with glutamic acid, at position 359 withasparagine, and at position 366 with serine (MgFc13); or a substitutionat position 316 with aspartic acid, at position 378 with valine, and atposition 399 with glutamic acid (MgFc27); or a substitution at position243 with isoleucine, at position 379 with leucine, and at position 420with valine (MgFc29); or a substitution at position 392 with threonineand at position 396 with leucine (MgFc38); or a substitution at position221 with glutamic acid, at position 270 with glutamic acid, at position308 with alanine, at position 311 with histidine, at position 396 withleucine, and at position 402 with aspartic (MgFc42); or a substitutionat position 410 with histidine, and at position 396 with leucine(MgFc53); or a substitution at position 243 with leucine, at position305 with isoleucine, at position 378 with aspartic acid, at position 404with serine, and at position 396 with leucine (MgFc54); or asubstitution at position 255 with isoleucine, and at position 396 withleucine (MgFc55); or a substitution at position 370 with glutamic acid,and at position 396 with leucine (MgFc59); or a substitution at position243 with leucine, at position 292 with proline, at position 300 withleucine, at position 305 with isoleucine, and at position 396 withleucine (MgFc88); or a substitution at position 243 with leucine, atposition 292 with proline, at position 300 with leucine, and at position396 with leucine (MgFc88A); or a substitution at position 234 withleucine, at position 292 with proline, and at position 300 with leucine(MgFc155); or a substitution at position 243 with leucine, at position292 with proline, and at position 300 with leucine; or a substitution atposition 243 with leucine, at position 292 with proline, and at position396 with leucine; or a substitution at position 243 with leucine, and atposition 292 with proline; or a substitution at position 243 withleucine; or a substitution at position 273 with phenylalanine.

6.1.6 Polypeptide and Antibody Conjugates

Molecules of the invention (i.e., polypeptides, antibodies) comprisingvariant Fc regions may be recombinantly fused or chemically conjugated(including both covalently and non-covalently conjugations) toheterologous polypeptides (i.e., an unrelated polypeptide; or portionthereof, preferably at least 10, at least 20, at least 30, at least 40,at least 50, at least 60, at least 70, at least 80, at least 90 or atleast 100 amino acids of the polypeptide) to generate fusion proteins.The fusion does not necessarily need to be direct, but may occur throughlinker sequences.

Further, molecules of the invention (i.e., polypeptides, antibodies)comprising variant Fc regions may be conjugated to a therapeutic agentor a drug moiety that modifies a given biological response. Therapeuticagents or drug moieties are not to be construed as limited to classicalchemical therapeutic agents. For example, the drug moiety may be aprotein or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin, gelonin,and pokeweed antiviral protein, a protein such as tumor necrosis factor,interferons including, but not limited to, α-interferon (IFN-α),β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent(e.g., TNF-α, TNF-β, AIM I as disclosed in PCT Publication No. WO97/33899), AIM II (see, PCT Publication No. WO 97/34911), Fas Ligand(Takahashi et al., J. Immunol., 6:1567-1574, 1994), and VEGI (PCTPublication No. WO 99/23105), a thrombotic agent or an anti-angiogenicagent (e.g., angiostatin or endostatin), or a biological responsemodifier such as, for example, a lymphokine (e.g., interleukin-1(“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocytemacrophage colony stimulating factor (“GM-CSF”), and granulocyte colonystimulating factor (“G-CSF”), macrophage colony stimulating factor,(“M-CSF”), or a growth factor (e.g., growth hormone (“GH”); proteases,or ribonucleases.

Molecules of the invention (i.e., polypeptides, antibodies) can be fusedto marker sequences, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., 1989, Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell,37:767 1984) and the “flag” tag (Knappik et al. Biotechniques,17(4):754-761, 1994).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of molecules of the invention (e.g.,antibodies with higher affinities and lower dissociation rates). See,generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol. 16:76; Hansson, et al.,1999, J. Mol. Biol. 287:265; and Lorenzo and Blasco, 1998, BioTechniques24:308 (each of these patents and publications are hereby incorporatedby reference in its entirety). Molecules of the invention comprisingvariant Fc regions, or the nucleic acids encoding the molecules of theinvention, may be further altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. One or more portions of a polynucleotideencoding a molecule of the invention, may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

The present invention also encompasses molecules of the inventioncomprising variant Fc regions (i.e., antibodies, polypeptides)conjugated to a diagnostic or therapeutic agent or any other moleculefor which serum half-life is desired to be increased and/or targeted toa particular subset of cells. The molecules of the invention can be useddiagnostically to, for example, monitor the development or progressionof a disease, disorder or infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the molecules of the inventionto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to themolecules of the invention or indirectly, through an intermediate (suchas, for example, a linker known in the art) using techniques known inthe art. See, for example, U.S. Pat. No. 4,741,900 for metal ions whichcan be conjugated to antibodies for use as diagnostics according to thepresent invention. Such diagnosis and detection can be accomplished bycoupling the molecules of the invention to detectable substancesincluding, but not limited to, various enzymes, enzymes including, butnot limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic group complexessuch as, but not limited to, streptavidin/biotin and avidin/biotin;fluorescent materials such as, but not limited to, umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent material such as, but not limited to, luminol;bioluminescent materials such as, but not limited to, luciferase,luciferin, and aequorin; radioactive material such as, but not limitedto, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co),fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm) rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), ruthenium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

Molecules of the invention (i.e., antibodies, polypeptides) comprising avariant Fc region may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine(CCNU), cyclophosphamide busulfan, dibromomannitol, streptozotocin,mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents(e.g., vincristine and vinblastine).

Moreover, a molecule of the invention can be conjugated to therapeuticmoieties such as a radioactive materials or macrocyclic chelators usefulfor conjugating radiometal ions (see above for examples of radioactivematerials). In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each of which is incorporated herein by reference in their entireties.

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol. Rev.,62:119-58, 1982.

In one embodiment, where the molecule of the invention is an antibodycomprising a variant Fc region, it can be administered with or without atherapeutic moiety conjugated to it, administered alone, or incombination with cytotoxic factor(s) and/or cytokine(s) for use as atherapeutic treatment. Alternatively, an antibody of the invention canbe conjugated to a second antibody to form an antibody heteroconjugateas described by Segal in U.S. Pat. No. 4,676,980, which is incorporatedherein by reference in its entirety. Antibodies of the invention mayalso be attached to solid supports, which are particularly useful forimmunoassays or purification of the target antigen. Such solid supportsinclude, but are not limited to, glass, cellulose, polyacrylamide,nylon, polystyrene, polyvinyl chloride or polypropylene.

6.2 Screening of Molecules with Variant Fc Regions for Enhanced FcγRIIIBinding and Characterization of Same

In preferred embodiments, screening and identifying molecules comprisingvariant Fc regions with altered FcγR affinities (e.g., enhanced FcγRIIIAaffinity) are done using the yeast display technology in combinationwith one or more biochemical based assays, preferably in a highthroughput manner, as described herein or as disclosed in U.S PatentApplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351, each of whichis hereby incorporated by reference in its entirety. The one or morebiochemical assays can be any assay known in the art for identifyingFc-FcγR interaction, i.e., specific binding of an Fc region to an FcγR,including, but not limited to, an ELISA assay, surface plasmon resonanceassays, immunoprecipitation assay, affinity chromatography, andequilibrium dialysis. In some embodiments, screening and identifyingmolecules comprising variant Fc regions with altered FcγR affinities(e.g., enhanced FcγRIIIA affinity) are done using the yeast displaytechnology as described herein in combination with one or morefunctional based assays, preferably in a high throughput manner. Thefunctional based assays can be any assay known in the art forcharacterizing one or more FcγR mediated effector cell functions such asthose described herein in Section 6.2.7. Non-limiting examples ofeffector cell functions that can be used in accordance with the methodsof the invention, include but are not limited to, antibody-dependentcell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis,phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting,C1q binding, and complement dependent cell mediated cytotoxicity. Insome embodiments, screening and identifying molecules comprising variantFc regions with altered FcγR affinities (e.g., enhanced FcγRIIIAaffinity) are done using the yeast display technology as describedherein in combination with one or more biochemical based assays incombination or in parallel with one or more functional based assays,preferably in a high throughput manner.

The term “specific binding” of an Fc region to an FcγR refers to aninteraction of the Fc region and a particular FcγR which has an affinityconstant of at least about 150 nM, in the case of monomeric FcγRIIIA andat least about 60 nM in the case of dimeric FcγRIIB as determined using,for example, an ELISA or surface plasmon resonance assay (e.g., aBIAcore™). The affinity constant of an Fc region for monomeric FcγRIIIAmay be 150 nM, 200 nM or 300 nM. The affinity constant of an Fc regionfor dimeric FcγRIIB may be 60 nM, 80 nM, 90 nM, or 100 nM. DimericFcγRIIB for use in the methods of the invention may be generated usingmethods known to one skilled in the art. Typically, the extracellularregion of FcγRIIB is covalently linked to a heterologous polypeptidewhich is capable of dimerization, so that the resulting fusion proteinis a dimer, e.g., see, U.S. Application No. 60/439,709 filed on Jan. 13,2003, which is incorporated herein by reference in its entirety. Aspecific interaction generally is stable under physiological conditions,including, for example, conditions that occur in a living individualsuch as a human or other vertebrate or invertebrate, as well asconditions that occur in a cell culture such conditions as used formaintaining and culturing mammalian cells or cells from anothervertebrate organism or an invertebrate organism.

In a specific embodiment, screening for and identifying moleculescomprising variant Fc regions and altered FcγR affinities comprise:displaying the molecule comprising a variant Fc region on the yeastsurface; and characterizing the binding of the molecule comprising thevariant Fc region to a FcγR (one or more), using a biochemical assay fordetermining Fc-FcγR interaction, preferably, an ELISA based assay. Oncethe molecule comprising a variant Fc region has been characterized forits interaction with one or more FcγRs and determined to have an alteredaffinity for one or more FcγRs, by at least one biochemical based assay,e.g., an ELISA assay, the molecule may be engineered into a completeimmunoglobulin, using standard recombinant DNA technology methods knownin the art, and the immunoglobulin comprising the variant Fc regionexpressed in mammalian cells for further biochemical characterization.The immunoglobulin into which a variant Fc region of the invention isintroduced (e.g., replacing the Fc region of the immunoglobulin) can beany immunoglobulin including, but not limited to, polyclonal antibodies,monoclonal antibodies, bispecific antibodies, multi-specific antibodies,humanized antibodies, and chimeric antibodies. In preferred embodiments,a variant Fc region is introduced into an immunoglobulin specific for acell surface receptor, a tumor antigen, or a cancer antigen. Theimmunoglobulin into which a variant Fc region of the invention isintroduced may specifically bind a cancer or tumor antigen for example,including, but not limited to, KS ¼ pan-carcinoma antigen (Perez andWalker, 1990, J. Immunol. 142: 3662-3667; Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991, CancerRes. 51(2): 468-475), prostatic acid phosphate (Tailor et al., 1990,Nucl. Acids Res. 18(16): 4928), prostate specific antigen (Henttu andVihko, 1989, Biochem. Biophys. Res. Comm. 160(2): 903-910; Israeli etal., 1993, Cancer Res. 53: 227-230), melanoma-associated antigen p97(Estin et al., 1989, J. Natl. Cancer Instit. 81(6): 445-446), melanomaantigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali etal., 1987, Cancer 59: 55-63; Mittelman et al., 1990, J. Clin. Invest.86: 2136-2144), prostate specific membrane antigen, carcinoembryonicantigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13: 294),polymorphic epithelial mucin antigen, human milk fat globule antigen,colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokata etal., 1992, Cancer Res. 52: 3402-3408), CO17-1A (Ragnhammar et al., 1993,Int. J. Cancer 53: 751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin.Immunol. 2: 135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19(Ghetie et al., 1994, Blood 83: 1329-1336), human B-lymphomaantigen-CD20 (Reff et al., 1994, Blood 83:435-445), CD33 (Sgouros etal., 1993, J. Nucl. Med. 34:422-430), melanoma specific antigens such asganglioside GD2 (Saleh et al., 1993, J. Immunol., 151, 3390-3398),ganglioside GD3 (Shitara et al., 1993, Cancer Immunol. Immunother.36:373-380), ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol.12: 1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250), tumor-specific transplantation type of cell-surface antigen(TSTA) such as virally-induced tumor antigens including T-antigen DNAtumor viruses and Envelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46: 3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245: 301-304), differentiation antigen (Feizi,1985, Nature 314: 53-57) such as I antigen found in fetal erythrocytes,primary endoderm I antigen found in adult erythrocytes, preimplantationembryos, I(Ma) found in gastric adenocarcinomas, M18, M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl,VIM-D5, D₁56-22 found in colorectal cancer, TRA-1-85 (blood group H),C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma,AH6 found in gastric cancer, Y hapten, Le^(y) found in embryonalcarcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells,E₁ series (blood group B) found in pancreatic cancer, FC10.2 found inembryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (bloodgroup Le^(a)) found in Adenocarcinoma, NS-10 found in adenocarcinomas,CO-43 (blood group Le^(b)), G49 found in EGF receptor of A431 cells, MH2(blood group ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 foundin colon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2), andM1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos. In one embodiment, the antigen is aT cell receptor derived peptide from a Cutaneous Tcell Lymphoma (see,Edelson, 1998, The Cancer Journal 4:62).

The invention particularly concerns the embodiment in which the bindingof the Fc variant to an FcγR activates a cellular effector which targetscells that array a cancer antigen such as A33 (a colorectal carcinomaantigen; Almqvist, Y. 2006, Nucl Med Biol. Nov.; 33(8):991-998); B1(Egloff, A. M. et al. 2006, Cancer Res. 66(1):6-9); BAGE (Bodey, B. 2002Expert Opin Biol Ther. 2(6):577-84); beta-catenin (Prange W. et al. 2003J Pathol. 201(2):250-9); CA125 (Bast, R. C. Jr. et al. 2005 Int JGynecol Cancer 15 Suppl 3:274-81); CD5 (Calin, G. A. et al. 2006 SeminOncol. 33(2):167-73; CD19 (Troussard, X. et al. 1998 Hematol Cell Ther.40(4):139-48); CD20 (Thomas, D. A. et al. 2006 Hematol Oncol Clin NorthAm. 20(5):1125-36); CD22 (Kreitman, R. J. 2006 AAPS J. 18;8(3):E532-51); CD23 (Rosati, S. et al. 2005 Curr Top Microbiol Immunol.5; 294:91-107); CD25 (Troussard, X. et al. 1998 Hematol Cell Ther.40(4):139-48); CD27 (Bataille, R. 2006 Haematologica 91(9):1234-40);CD28 (Bataille, R. 2006 Haematologica 91(9):1234-40); CD36 (Ge, Y. 2005Lab Hematol. 11(1):31-7); CD40/CD154 (Messmer, D. et al. 2005 Ann N YAcad Sci. 1062:51-60); CD45 (Jurcic, J. G. 2005 Curr Oncol Rep.7(5):339-46); CD56 (Bataille, R. 2006 Haematologica 91(9):1234-40);CD79a/CD79b (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48;Chu, P. G. et al. 2001 Appl Immunohistochem Mol. Morphol. 9(2):97-106);CD103 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CDK4(Lee, Y. M. et al. 2006 Cell Cycle 5(18):2110-4); CEA (carcinoembryonicantigen; Mathelin, C. 2006 Gynecol Obstet Fertil. 34(7-8):638-46;Tellez-Avila, F. I. et al. 2005 Rev Invest Clin. 57(6):814-9); CTLA4(Peggs, K. S. et al. 2006 Curr Opin Immunol. 18(2):206-13); EGF-R(epidermal growth factor receptor; Adenis, A. et al. 2003 Bull Cancer.90 Spec No: S228-32); Erb (ErbB1; ErbB3; ErbB4; Zhou, H. et al. 2002Oncogene 21(57):8732-40; Rimon, E. et al. 2004 Int J Oncol.24(5):1325-38); GAGE (GAGE-1; GAGE-2; Akcakanat, A. et al. 2006 Int JCancer. 118(1):123-8); GD2/GD3/GM2 (Livingston, P. O. et al. 2005 CancerImmunol Immunother. 54(10):1018-25); gp100 (Lotem, M. et al. 2006 JImmunother. 29(6):616-27); HER-2/neu (Kumar, Pal S et al. 2006 SeminOncol. 33(4):386-91); human papillomavirus-E6/human papillomavirus-E7(DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; KSA (17-1A)(Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); MAGE (MAGE-1; MAGE-3;(Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); MART (Kounalakis,N. et al. 2005 Curr Oncol Rep. 7(5):377-82; MUC-1 (Mathelin, C. 2006Gynecol Obstet. Fertil. 34(7-8):638-46); MUM-1 (Castelli, C. et al. 2000J Cell Physiol. 182(3):323-31); N-acetylglucosaminyltransferase (Dennis,J. W. 1999 Biochim Biophys Acta. 6; 1473(1):21-34); p15 (Gil, J. et al.2006 Nat Rev Mol Cell Biol. 7(9):667-77); PSA (prostate specificantigen; Cracco, C. M. et al. 2005 Minerva Urol Nefrol. 57(4):301-11);PSMA (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); sTn (Holmberg,L. A. 2001 Expert Opin Biol Ther. 1(5):881-91); TNF-receptor (TNF-αreceptor, TNF-β receptor; or TNF-γ receptor; van Horssen, R. et al. 2006Oncologist. 11(4):397-408; Gardnerova, M. et al. 2000 Curr Drug Targets.1(4):327-64); or VEGF receptor (O'Dwyer. P. J. 2006 Oncologist.11(9):992-8). Also of interest are antigens specific to particularinfectious agents, e.g., viral agents including, but not limited tohuman immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza,human papilloma virus (HPV), foot and mouth (coxsackieviruses), therabies virus, herpes simplex virus (HSV), and the causative agents ofgastroenteritis, including rotaviruses, adenoviruses, caliciviruses,astroviruses and Norwalk virus; bacterial agents including, but notlimited to E. coli, Salmonella thyphimurium, Pseudomonas aeruginosa,Vibrio cholerae, Neisseria gonorrhoeae, Helicobacter pylori, Hemophilusinfluenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacteriumtuberculosis and Streptococcus pneumoniae, fungal agents and parasitessuch as Giardia.

In some embodiments, a variant Fc region of the invention is introducedinto an anti-fluoresceine monoclonal antibody, 4-4-20 (Kranz et al.,1982 J. Biol. Chem. 257(12): 6987-6995; which is incorporated herein byreference in its entirety). In other embodiments, a variant Fc region ofthe invention is introduced into a mouse-human chimeric anti-CD20monoclonal antibody 2H7, which recognizes the CD20 cell surfacephosphoprotein on B cells (Liu et al., 1987, Journal of Immunology, 139:3521-6; which is incorporated herein by reference in its entirety). Inyet other embodiments, a variant Fc region of the invention isintroduced into a humanized antibody (Ab4D5) against the human epidermalgrowth factor receptor 2 (p185 HER2) as described by Carter et al.(1992, Proc. Natl. Acad. Sci. USA 89: 4285-9; which is incorporatedherein by reference in its entirety). In yet other embodiments, avariant Fc region of the invention is introduced into a humanizedanti-TAG72 antibody (CC49) (Sha et al., 1994 Cancer Biother. 9(4):341-9). In other embodiments, a variant Fc region of the invention isintroduced into Rituxan which is used for treating lymphomas.

In another specific embodiment, the invention encompasses engineering ananti-FcγRIIB antibody including but not limited to any of the antibodiesdisclosed in U.S. Patent Application Publications 2005/02157667;2004/0185045; 2005/0260213; or 2006/013810; International PatentApplication Publications WO 2005/110474 or WO 2005/115452; U.S. patentapplication Ser. No. 11/305,787 filed Dec. 15, 2005; or ProvisionalApplications No. 60/809,116; 60/816,126; or 60/816,688 filed on May 26,2006, Jun. 23, 2006, or Jun. 26, 2006, respectively by modification(e.g., substitution, insertion, deletion) of at least one amino acidresidue which modification increases the affinity of the Fc region forFcγRIIIA and/or FcγRIIA. Each of the above mentioned references ishereby incorporated by reference in their entirety. Examples ofanti-FcγRIIB antibodies, which may or may not be humanized, that may beengineered in accordance with the methods of the invention are 2B6monoclonal antibody having ATCC accession number PTA-4591 and 3H7 havingATCC accession number PTA-4592, 1D5 monoclonal antibody having ATCCaccession number PTA-5958, 1F2 monoclonal antibody having ATCC accessionnumber PTA-5959, 2D11 monoclonal antibody having ATCC accession numberPTA-5960, 2E1 monoclonal antibody having ATCC accession number PTA-5961and 2H9 monoclonal antibody having ATCC accession number PTA-5962 (alldeposited at 10801 University Boulevard, Manassas, Va. 02209-2011),which are incorporated herein by reference. In another specificembodiment, modification of the anti-FcγRIIB antibody may also furtherdecrease the affinity of the Fc region for FcγRIIB. In yet anotherspecific embodiment, the engineered anti-FcγRIIB antibody may furtherhave an enhanced effector function as determined by standard assaysknown in the art and disclosed and exemplified herein. In someembodiments, a variant Fc region of the invention is introduced into atherapeutic monoclonal antibody specific for a cancer antigen or cellsurface receptor including but not limited to, Erbitux™ (also known asIMC-C225) (ImClone Systems Inc.), a chimerized monoclonal antibodyagainst EGFR; HERCEPTIN® (Trastuzumab) (Genentech, Calif.) which is ahumanized anti-HER2 monoclonal antibody for the treatment of patientswith metastatic breast cancer; REOPRO® (abciximab) (Centocor) which isan anti-glycoprotein IIb/IIIa receptor on the platelets for theprevention of clot formation; ZENAPAX® (daclizumab) (RochePharmaceuticals, Switzerland) which is an immunosuppressive, humanizedanti-CD25 monoclonal antibody for the prevention of acute renalallograft rejection. Other examples are a humanized anti-CD18 F(ab′)₂(Genentech); CDP860 which is a humanized anti-CD 18 F(ab′)₂ (Celltech,UK); PRO542 which is an anti-HIV gp120 antibody fused with CD4(Progenics/Genzyme Transgenics); C14 which is an anti-CD14 antibody(ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech); OVAREX™which is a murine anti-CA 125 antibody (Altarex); PANOREX™ which is amurine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); IMC-C225 which is a chimeric anti-EGFR IgG antibody(ImClone System); VITAXIN™ which is a humanized anti-αVβ3 integrinantibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03which is a humanized anti CD52 IgG1 antibody (Leukosite); Smart M195which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo);RITUXAN™ which is a chimeric anti-CD20 IgG1 antibody (IDECPharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanizedanti-HLA antibody (Protein Design Lab); ONCOLYM™ (Lym-1) is aradiolabelled murine anti-HLA DR antibody (Techniclone); anti-CD11a is ahumanized IgG1 antibody (Genetech/Xoma); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm);IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKlineBeecham); MDX-CD4 is a human anti-CD4 IgG antibody(Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α. IgG4 antibody(Celltech); LDP-02 is a humanized anti-α4β7 antibody(LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgGantibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgG antibody(Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody (Elan);MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);rhuMab-E25 is a humanized anti-IgE IgG1 antibody(Genentech/Norvartis/Tanox Biosystems); IDEC-152 is a primatizedanti-CD23 antibody (IDEC Pharm); ABX-CBL is a murine anti CD-147 IgMantibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′)₂(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor).

The variant Fc regions of the invention, preferably in the context of animmunoglobulin, can be further characterized using one or morebiochemical assays and/or one or more functional assays, preferably in ahigh throughput manner. In some alternate embodiments, the variant Fcregions of the inventions are not introduced into an immunoglobulin andare further characterized using one or more biochemical based assaysand/or one or more functional assays, preferably in a high throughputmanner. The one or more biochemical assays can be any assay known in theart for identifying Fc-FcγR interactions, including, but not limited to,an ELISA assay, and surface plasmon resonance-based assay fordetermining the kinetic parameters of Fc-FcγR interaction, e.g., BIAcoreassay. The one or more functional assays can be any assay known in theart for characterizing one or more FcγR mediated effector cell functionas known to one skilled in the art or described herein. In specificembodiments, the immunoglobulins comprising the variant Fc regions areassayed in an ELISA assay for binding to one or more FcγRs, e.g.,FcγRIIIA, FcγRIIA, FcγRIIA; followed by one or more ADCC assays. In someembodiments, the immunoglobulins comprising the variant Fc regions areassayed further using a surface plasmon resonance-based assay, e.g.,BIAcore. Surface plasmon resonance-based assays are well known in theart, and are further discussed in Section 6.2.7, and exemplified hereinin Example 7.8.

An exemplary high throughput assay for characterizing immunoglobulinscomprising variant Fc regions may comprise: introducing a variant Fcregion of the invention, e.g., by standard recombinant DNA technologymethods, in a 4-4-20 antibody; characterizing the specific binding ofthe 4-4-20 antibody comprising the variant Fc region to an FcγR (e.g.,FcγRIIIA, FcγRIIB) in an ELISA assay; characterizing the 4-4-20 antibodycomprising the variant Fc region in an ADCC assay (using methodsdisclosed herein) wherein the target cells are opsonized with the 4-4-20antibody comprising the variant Fc region; the variant Fc region maythen be cloned into a second immunoglobulin, e.g., 4D5, 2H7, and thatsecond immunoglobulin characterized in an ADCC assay, wherein the targetcells are opsonized with the second antibody comprising the variant Fcregion. The second antibody comprising the variant Fc region is thenfurther analyzed using an ELISA-based assay to confirm the specificbinding to an FcγR.

Preferably, a variant Fc region of the invention binds FcγRIIIA and/orFcγRIIA with a higher affinity than a wild type Fc region as determinedin an ELISA assay. Most preferably, a variant Fc region of the inventionbinds FcγRIIIA and/or FcγRIIA with a higher affinity and binds FcγRIIBwith a lower affinity than a wild type Fc region as determined in anELISA assay. In some embodiments, the variant Fc region binds FcγRIIIAand/or FcγRIIA with at least 2-fold higher, at least 4-fold higher, morepreferably at least 6-fold higher, most preferably at least 8 to 10-foldhigher affinity than a wild type Fc region binds FcγRIIIA and/or FcγRIIAand binds FcγRIIB with at least 2-fold lower, at least 4-fold lower,more preferably at least 6-fold lower, most preferably at least 8 to10-fold lower affinity than a wild type Fc region binds FcγRIIB asdetermined in an ELISA assay.

The immunoglobulin comprising the variant Fc regions may be analyzed atany point using a surface plasmon based resonance based assay, e.g.,BIAcore, for defining the kinetic parameters of the Fc-FcγR interaction,using methods disclosed herein and known to those of skill in the art.Preferably, the Kd of a variant Fc region of the invention for bindingto a monomeric FcγRIIIA and/or FcγRIIA as determined by BIAcore analysisis about 100 nM, preferably about 70 nM, most preferably about 40 nM.;and the Kd of the variant Fc region of the invention for binding adimeric FcγRIIB is about 80 nM, about 100 nM, more preferably about 200nM.

In most preferred embodiments, the immunoglobulin comprising the variantFc regions is further characterized in an animal model for interactionwith an FcγR. Preferred animal models for use in the methods of theinvention are, for example, transgenic mice expressing human FcγRs,e.g., any mouse model described in U.S. Pat. Nos. 5,877,397, and6,676,927 which are incorporated herein by reference in their entirety.Transgenic mice for use in the methods of the invention include, but arenot limited to, nude knockout FcγRIIIA mice carrying human FcγRIIIA;nude knockout FcγRIIIA mice carrying human FcγRIIA; nude knockoutFcγRIIIA mice carrying human FcγRIIB and human FcγRIIIA; nude knockoutFcγRIIIA mice carrying human FcγRIIB and human FcγRIIA; nude knockoutFcγRIIIA and FcγRIIA mice carrying human FcγRIIIA and FcγRIIA and nudeknockout FcγRIIIA, FcγRIIA and FcγRIIB mice carrying human FcγRIIIA,FcγRIIA and FcγRIIB.

6.2.1 Design Strategies

The present invention encompasses engineering methods to generate Fcvariants including but not limited to computational design strategies,library generation methods, and experimental production and screeningmethods. These strategies may be applied individually or in variouscombinations to engineer the Fc variants of the instant invention.

In most preferred embodiments, the engineering methods of the inventioncomprise methods in which amino acids at the interface between an Fcregion and the Fc ligand are not modified. Fc ligands include but arenot limited to FcγRs, C1q, FcRn, C3, mannose receptor, protein A,protein G, mannose receptor, and undiscovered molecules that bind Fc.Amino acids at the interface between an Fc region and an Fc ligand isdefined as those amino acids that make a direct and/or indirect contactbetween the Fc region and the ligand, play a structural role indetermining the conformation of the interface, or are within at least 3angstroms, preferably at least 2 angstroms of each other as determinedby structural analysis, such as x-ray crystallography and molecularmodeling. The amino acids at the interface between an Fc region and anFc ligand include those amino acids that make a direct contact with anFcγR based on crystallographic and structural analysis of Fc-FcγRinteractions such as those disclosed by Sondermann et al., (2000,Nature, 406: 267-273; which is incorporated herein by reference in itsentirety). Examples of positions within the Fc region that make a directcontact with FcγR are amino acids 234-239 (hinge region), amino acids265-269 (B/C loop), amino acids 297-299 (C′/E loop), and amino acids327-332 (F/G) loop. In some embodiments, the molecules of the inventioncomprising variant Fc regions comprise modification of at least oneresidue that does not make a direct contact with an FcγR based onstructural and crystallographic analysis, e.g., is not within theFc-FcγR binding site.

Preferably, the engineering methods of the invention do not modify anyof the amino acids as identified by Shields et al., which are located inthe CH2 domain of an Fc region proximal to the hinge region, e.g.,Leu234-Pro238; Ala327, Pro329, and affect binding of an Fc region to allhuman FcγRs.

In other embodiments, the invention encompasses Fc variants with alteredFcγR affinities and/or altered effector functions, such that the Fcvariant does not have an amino acid modification at a position at theinterface between an Fc region and the Fc ligand. Preferably, such Fcvariants in combination with one or more other amino acid modificationswhich are at the interface between an Fc region and the Fc ligand have afurther impact on the particular altered property, e.g. altered FcγRaffinity. Modifying amino acids at the interface between Fc and an Fcligand may be done using methods known in the art, for example based onstructural analysis of Fc-ligand complexes. For example but not by wayof limitation by exploring energetically favorable substitutions at Fcpositions that impact the binding interface, variants can be engineeredthat sample new interface conformations, some of which may improvebinding to the Fc ligand, some of which may reduce Fc ligand binding,and some of which may have other favorable properties. Such newinterface conformations could be the result of, for example, directinteraction with Fc ligand residues that form the interface, or indirecteffects caused by the amino acid modifications such as perturbation ofside chain or backbone conformations

The invention encompasses engineering Fc variants comprising any of theamino acid modifications disclosed herein in combination with othermodifications in which the conformation of the Fc carbohydrate atposition 297 is altered. The invention encompasses conformational andcompositional changes in the N297 carbohydrate that result in a desiredproperty, for example increased or reduced affinity for an FcγR. Suchmodifications may further enhance the phenotype of the original aminoacid modification of the Fc variants of the invention. Although notintending to be bound by a particular mechanism of actions such astrategy is supported by the observation that the carbohydrate structureand conformation dramatically affect Fc-FcγR and Fc/C1q binding (Umahaet al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, BiotechnolBioeng 74:288-294; Mimura et al., 2001, J Biol Chem 276:45539; Radaev etal., 2001, J Biol Chem 276:16478-16483; Shields et al. 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473).

Another design strategy for generating Fc variants in accordance withthe invention is provided in which the Fc region is reengineered toeliminate the structural and functional dependence on glycosylation.This design strategy involves the optimization of Fc structure,stability, solubility, and/or Fc function (for example affinity of Fcfor one or more Fc ligands) in the absence of the N297 carbohydrate. Inone approach, positions that are exposed to solvent in the absence ofglycosylation are engineered such that they are stable, structurallyconsistent with Fc structure, and have no tendency to aggregate.Approaches for optimizing aglycosylated Fc may involve but are notlimited to designing amino acid modifications that enhance aglycoslatedFc stability and/or solubility by incorporating polar and/or chargedresidues that face inward towards the Cg2-Cg2 dimer axis, and bydesigning amino acid modifications that directly enhance theaglycosylated Fc-FcγR interface or the interface of aglycosylated Fcwith some other Fc ligand.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that altereffector function. The invention encompasses combining an Fc variant ofthe invention with other Fc modifications to provide additive,synergistic, or novel properties in antibodies or Fc fusions. Suchmodifications may be in the CH1, CH2, or CH3 domains or a combinationthereof. Preferably the Fc variants of the invention enhance theproperty of the modification with which they are combined. For example,if an Fc variant of the invention is combined with a mutant known tobind FcγRIIIA with a higher affinity than a comparable moleculecomprising a wild type Fc region; the combination with a mutant of theinvention results in a greater fold enhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:49634969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, JImmunol 164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573;U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of whichis incorporated herein by reference in its entirety.

6.2.2 Functional Assays of Molecules with Variant Fc Regions

The invention encompasses characterization of the molecules of theinvention (e.g., an antibody comprising a variant Fc region identifiedby the yeast display technology and FcγR-Fc binding assays disclosed inU.S Patent Application Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351 (each of whichis hereby incorporated by reference in its entirety); or therapeuticmonoclonal antibodies engineered according to the methods of theinvention) using assays known to those skilled in the art foridentifying the effector cell function of the molecules. In particular,the invention encompasses characterizing the molecules of the inventionfor FcγR-mediated effector cell function. Examples of effector cellfunctions that can be assayed in accordance with the invention, includebut are not limited to, antibody-dependent cell mediated cytotoxicity,phagocytosis, opsonization, opsonophagocytosis, C1q binding, andcomplement dependent cell mediated cytotoxicity. Any cell-based or cellfree assay known to those skilled in the art for determining effectorcell function activity can be used (For effector cell assays, seePerussia et al., 2000, Methods Mol. Biol. 121: 179-92; Baggiolini etal., 1998 Experientia, 44(10): 841-8; Lehmann et al., 2000 J. Immunol.Methods, 243(1-2): 229-42; Brown E J. 1994, Methods Cell Biol., 45:147-64; Munn et al., 1990 J. Exp. Med., 172: 231-237, Abdul-Majid etal., 2002 Scand. J. Immunol. 55: 70-81; Ding et al., 1998, Immunity8:403-411, each of which is incorporated by reference herein in itsentirety).

In one embodiment, the molecules of the invention can be assayed forFcγR-mediated phagocytosis in human monocytes. Alternatively, theFcγR-mediated phagocytosis of the molecules of the invention may beassayed in other phagocytes, e.g., neutrophils (polymorphonuclearleuckocytes; PMN); human peripheral blood monocytes, monocyte-derivedmacrophages, which can be obtained using standard procedures known tothose skilled in the art (e.g., see Brown E J. 1994, Methods Cell Biol.,45: 147-164). In one embodiment, the function of the molecules of theinvention is characterized by measuring the ability of THP-1 cells tophagocytose fluoresceinated IgG-opsonized sheep red blood cells (SRBC)by methods previously described (Tridandapani et al. 2000, J. Biol.Chem. 275: 20480-7). For example, an exemplary assay for measuringphagocytosis of the molecules of the invention comprising variant Fcregions with enhanced affinities for FcγRIIIA, comprises of: treatingTHP-1 cells with a molecule of the invention or with a control antibodythat does not bind to FcγRIIIA, comparing the activity levels of saidcells, wherein a difference in the activities of the cells (e.g.,rosetting activity (the number of THP-1 cells binding IgG-coated SRBC),adherence activity (the total number of SRBC bound to THP-1 cells), andphagocytic rate) would indicate the functionality of the molecule of theinvention. It can be appreciated by one skilled in the art that thisexemplary assay can be used to assay any of the molecules identified bythe methods of the invention.

Another exemplary assay for determining the phagocytosis of themolecules of the invention is an antibody-dependent opsonophagocytosisassay (ADCP) which can comprise the following: coating a targetbioparticle such as Escherichia coli-labeled FITC (Molecular Probes) orStaphylococcus aureus-FITC with (i) wild-type 4-4-20 antibody, anantibody to fluorescein (See Bedzyk et al., 1989, J. Biol. Chem, 264(3):1565-1569, which is incorporated herein by reference in its entirety),as the control antibody for FcγR-dependent ADCP; or (ii) 4-4-20 antibodyharboring the D265A mutation that knocks out binding to FcγRIII, as abackground control for FcγR-dependent ADCP (iii) 4-4-20 antibodycarrying variant Fc regions identified by the methods of the inventionand produced as exemplified in Example 7.6; and forming the opsonizedparticle; adding any of the osponized particles described (i-iii) toTHP-1 effector cells (a monocytic cell line available from ATCC) in a60:1 ratio to allow FcγR-mediated phagocytosis to occur; preferablyincubating the cells and E. coli-FITC/antibody at 37° C. for 1.5 hour;adding trypan blue after incubation (preferably at room temperature for2-3 min.) to the cells to quench the fluorescence of the bacteria thatare adhered to the outside of the cell surface without beinginternalized; transferring cells into a FACS buffer (e.g., 0.1%, BSA inPBS, 0.1%, sodium azide), analyzing the fluorescence of the THP1 cellsusing FACS (e.g., BD FACS Calibur). Preferably, the THP-1 cells used inthe assay are analyzed by FACS for expression of FcγR on the cellsurface. THP-1 cells express both CD32A and CD64. CD64 is a highaffinity FcγR that is blocked in conducting the ADCP assay in accordancewith the methods of the invention. The THP-1 cells are preferablyblocked with 100 μg/ml, soluble IgG1 or 10% human serum. To analyze theextent of ADCP, the gate is preferably set on THP-1 cells and medianfluorescence intensity is measured. The ADCP activity for individualmutants is calculated and reported as a normalized value to the wildtype chMab 4-4-20 obtained. The opsonized particles are added to THP-1cells such that the ratio of the opsonized particles to THP-1 cells is30:1 or 60:1. In most preferred embodiments, the ADCP assay is conductedwith controls, such as E. coli-FITC in medium, E. coli-FITC and THP-1cells (to serve as FcγR-independent ADCP activity), E. coli-FITC, THP-1cells and wild-type 4-4-20 antibody (to serve as FcγR-dependent ADCPactivity), E coli-FITC, THP-1 cells, 4-4-20 D265A (to serve as thebackground control for FcγR-dependent ADCP activity).

In another embodiment, the molecules of the invention can be assayed forFcγR-mediated ADCC activity in effector cells, e.g., natural killercells, using any of the standard methods known to those skilled in theart (See e.g., Perussia et al., 2000, Methods Mol. Biol. 121: 179-92).An exemplary assay for determining ADCC activity of the molecules of theinvention is based on a ⁵¹Cr release assay comprising of: labelingtarget cells with [⁵¹Cr]Na₂CrO₄ (this cell-membrane permeable moleculeis commonly used for labeling since it binds cytoplasmic proteins andalthough spontaneously released from the cells with slow kinetics, it isreleased massively following target cell necrosis); osponizing thetarget cells with the molecules of the invention comprising variant Fcregions; combining the opsonized radiolabeled target cells with effectorcells in a microtitre plate at an appropriate ratio of target cells toeffector cells; incubating the mixture of cells for 16-18 hours at 37°C.; collecting supernatants; and analyzing radioactivity. Thecytotoxicity of the molecules of the invention can then be determined,for example using the following formula: % lysis=(experimentalcpm−target leak cpm)/(detergent lysis cpm−target leak cpm)×100%.Alternatively, % lysis=(ADCC-AICC)/(maximum release-spontaneousrelease). Specific lysis can be calculated using the formula: specificlysis=% lysis with the molecules of the invention−% lysis in the absenceof the molecules of the invention. A graph can be generated by varyingeither the target:effector cell ratio or antibody concentration.

In yet another embodiment, the molecules of the invention arecharacterized for antibody dependent cellular cytotoxicity (ADCC) see,e.g., Ding et al., Immunity, 1998, 8:403-11; which is incorporatedherein by reference in its entirety.

Preferably, the effector cells used in the ADCC assays of the inventionare peripheral blood mononuclear cells (PBMC) that are preferablypurified from normal human blood, using standard methods known to oneskilled in the art, e.g., using Ficoll-Paque density gradientcentrifugation. Preferred effector cells for use in the methods of theinvention express different FcγR activating receptors. The inventionencompasses, effector cells, THP-1, expressing FcγRI, FcγRIIA andFcγRIIB, and monocyte derived primary macrophages derived from wholehuman blood expressing both FcγRIIIA and FcγRIIB, to determine if Fcantibody mutants show increased ADCC activity and phagocytosis relativeto wild type IgG1 antibodies.

The human monocyte cell line, THP-1, activates phagocytosis throughexpression of the high affinity receptor FcγRI and the low affinityreceptor FcγRIIA (Fleit et al., 1991, J. Leuk. Biol. 49: 556). THP-1cells do not constitutively express FcγRIIA or FcγRIIB. Stimulation ofthese cells with cytokines effects the FcR expression pattern (Pricop etal., 2000 J. Immunol. 166: 531-7). Growth of THP-1 cells in the presenceof the cytokine IL4 induces FcγRIIB expression and causes a reduction inFcγRIIA and FcγRI expression. FcγRIIB expression can also be enhanced byincreased cell density (Tridandapani et al., 2002, J. Biol Chem. 277:5082-9). In contrast, it has been reported that IFNγ can lead toexpression of FcγRIIIA (Pearse et al., 1993 PNAS USA 90: 4314-8). Thepresence or absence of receptors on the cell surface can be determinedby FACS using common methods known to one skilled in the art. Cytokineinduced expression of FcγR on the cell surface provides a system to testboth activation and inhibition in the presence of FcγRIIB. If THP-1cells are unable to express the FcγRIIB the invention also encompassesanother human monocyte cell line, U937. These cells have been shown toterminally differentiate into macrophages in the presence of IFNγ andTNF (Koren et al., 1979, Nature 279: 328-331).

FcγR dependent tumor cell killing is mediated by macrophage and NK cellsin mouse tumor models (Clynes et al., 1998, PNAS USA 95: 652-656). Theinvention encompasses the use of elutriated monocytes from donors aseffector cells to analyze the efficiency Fc mutants to trigger cellcytotoxicity of target cells in both phagocytosis and ADCC assays.Expression patterns of FcγRI, FcγRIIIA, and FcγRIIB are affected bydifferent growth conditions. FcγR expression from frozen elutriatedmonocytes, fresh elutriated monocytes, monocytes maintained in 10% FBS,and monocytes cultured in FBS+GM-CSF and or in human serum may bedetermined using common methods known to those skilled in the art. Forexample, cells can be stained with FcγR specific antibodies and analyzedby FACS to determine FcR profiles. Conditions that best mimic macrophagein vivo FcγR expression is then used for the methods of the invention.

In some embodiments, the invention encompasses the use of mouse cellsespecially when human cells with the right FcγR profiles are unable tobe obtained. In some embodiments, the invention encompasses the mousemacrophage cell line RAW264.7(ATCC) which can be transfected with humanFcγRIIIA and stable transfectants isolated using methods known in theart, see, e.g., Ralph et al., J. Immunol. 119: 950-4). Transfectants canbe quantitated for FcγRIIIA expression by FACS analysis using routineexperimentation and high expressors can be used in the ADCC assays ofthe invention. In other embodiments, the invention encompasses isolationof spleen peritoneal macrophage expressing human FcγR from knockouttransgenic mice such as those disclosed herein.

Lymphocytes may be harvested from peripheral blood of donors (PBM) usinga Ficoll-Paque gradient (Pharmacia). Within the isolated mononuclearpopulation of cells the majority of the ADCC activity occurs via thenatural killer cells (NK) containing FcγRIIIA but not FcγRIIB on theirsurface. Results with these cells indicate the efficacy of the mutantson triggering NK cell ADCC and establish the reagents to test withelutriated monocytes.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), and Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10). The target cells must be recognized by the antigen bindingsite of the immunoglobulin to be assayed.

The ADCC assay is based on the ability of NK cells to mediate cell deathvia an apoptotic pathway. NK cells mediate cell death in part byFcγRIIIA's recognition of IgG bound to an antigen on a cell surface. TheADCC assays used in accordance with the methods of the invention may beradioactive based assays or fluorescence based assays. The ADCC assayused to characterize the molecules of the invention comprising variantFc regions comprises labeling target cells, e.g., SK-BR-3, MCF-7,OVCAR3, Raji, Daudi cells, opsonizing target cells with an antibody thatrecognizes a cell surface receptor on the target cell via its antigenbinding site; combining the labeled opsonized target cells and theeffector cells at an appropriate ratio, which can be determined byroutine experimentation; harvesting the cells; detecting the label inthe supernatant of the lysed target cells, using an appropriatedetection scheme based on the label used. The target cells may belabeled either with a radioactive label or a fluorescent label, usingstandard methods known in the art. For example the labels include, butare not limited to, [⁵¹Cr]Na₂CrO₄; and the acetoxymethyl ester of thefluorescence enhancing ligand, 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate(TDA).

In a specific preferred embodiment, a time resolved fluorimetric assayis used for measuring ADCC activity against target cells that have beenlabeled with the acetoxymethyl ester of the fluorescence enhancingligand, 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate (TDA). Suchfluorimetric assays are known in the art, e.g., see, Blomberg et al.,1996, Journal of Immunological Methods, 193: 199-206; which isincorporated herein by reference in its entirety. Briefly, target cellsare labeled with the membrane permeable acetoxymethyl diester of TDA(bis(acetoxymethyl) 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate, (BATDA),which rapidly diffuses across the cell membrane of viable cells.Intracellular esterases split off the ester groups and the regeneratedmembrane impermeable TDA molecule is trapped inside the cell. Afterincubation of effector and target cells, e.g., for at least two hours,up to 3.5 hours, at 37° C., under 5% CO₂, the TDA released from thelysed target cells is chelated with Eu3+ and the fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallac).

In another specific embodiment, the ADCC assay used to characterize themolecules of the invention comprising variant Fc regions comprises thefollowing steps: Preferably 4−5×10⁶ target cells (e.g., SK-BR-3, MCF-7,OVCAR3, Raji cells) are labeled with bis(acetoxymethyl)2,2′:6′,2″-terpyridine-t-6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). For optimal labeling efficiency, the number of targetcells used in the ADCC assay should preferably not exceed 5×10⁶. BATDAreagent is added to the cells and the mixture is incubated at 37° C.preferably under 5% CO₂, for at least 30 minutes. The cells are thenwashed with a physiological buffer, e.g., PBS with 0.125 mMsulfinpyrazole, and media containing 0.125 mM sulfinpyrazole. Thelabeled target cells are then opsonized (coated) with a molecule of theinvention comprising a variant Fc region, i.e., an immunoglobulincomprising a variant Fc region of the invention, including, but notlimited to, a polyclonal antibody, a monoclonal antibody, a bispecificantibody, a multi-specific antibody, a humanized antibody, or a chimericantibody. In preferred embodiments, the immunoglobulin comprising avariant Fc region used in the ADCC assay is specific for a cell surfacereceptor, a tumor antigen, or a cancer antigen. The immunoglobulin intowhich a variant Fc region of the invention is introduced mayspecifically bind any cancer or tumor antigen, such as those listed inSection 6.4. Additionally, the immunoglobulin into which a variant Fcregion of the invention is introduced may be any therapeutic antibodyspecific for a cancer antigen, such as those listed in Section 6.4. Insome embodiments, the immunoglobulin comprising a variant Fc region usedin the ADCC assay is an anti-fluoresceine monoclonal antibody, 4-4-20(Kranz et al., 1982 J. Biol. Chem. 257(12): 6987-6995) a mouse-humanchimeric anti-CD20 monoclonal antibody 2H7 (Liu et al., 1987, Journal ofImmunology, 139: 3521-6); or a humanized antibody (Ab4D5) against thehuman epidermal growth factor receptor 2 (p185 HER2) (Carter et al.(1992, Proc. Natl. Acad. Sci. USA 89: 4285-9). The target cells in theADCC assay are chosen according to the immunoglobulin into which avariant Fc region of the invention has been introduced so that theimmunoglobulin binds a cell surface receptor of the target cellspecifically. Preferably, the ADCC assays of the invention are performedusing more than one engineered antibody, e.g., anti Her2/neu, 4-4-20,2B6, Rituxan, and 2H7, harboring the Fc variants of the invention. In amost preferred embodiment, the Fc variants of the invention areintroduced into at least 3 antibodies and their ADCC activities aretested. Although not intending to be bound by a particular mechanism ofaction, examining at least 3 antibodies in these functional assays willdiminish the chance of eliminating a viable Fc mutation erroneously.

Opsonized target cells are added to effector cells, e.g., PBMC, toproduce effector:target ratios of approximately 50:1, 75:1, or 100:1. Ina specific embodiment, when the immunoglobulin comprising a variant Fcregion has the variable domain of 4-4-20, the effector:target is 75:1.The effector and target cells are incubated for at least two hours, upto 3.5 hours, at 37° C., under 5% CO₂. Cell supernatants are harvestedand added to an acidic europium solution (e.g., DELFIA EuropiumSolution, Perkin Elmer/Wallac). The fluorescence of the Europium-TDAchelates formed is quantitated in a time-resolved fluorometer (e.g.Victor 1420, Perkin Elmer/Wallac). Maximal release (MR) and spontaneousrelease (SR) are determined by incubation of target cells with 1% TX-100and media alone, respectively. Antibody independent cellularcytotoxicity (AICC) is measured by incubation of target and effectorcells in the absence of antibody. Each assay is preferably performed intriplicate. The mean percentage specific lysis is calculated as:Experimental release(ADCC)−AICC)/(MR−SR)×100.

The invention encompasses characterization of the Fc variants in bothNK-dependent and macrophage dependent ADCC assays. Fc variants of theinvention have altered phenotypes such as an altered effector functionas assayed in an NK dependent or macrophage dependent assay.

The invention encompasses assays known in the art and exemplifiedherein, to bind C1q and mediate complement dependent cytotoxicity (CDC).To determine C1q binding, a C1q binding ELISA may be performed. Anexemplary assay may comprise the following: assay plates may be coatedovernight at 4 C with polypeptide variant or starting polypeptide(control) in coating buffer. The plates may then be washed and blocked.Following washing, an aliquot of human C1q may be added to each well andincubated for 2 hrs at room temperature. Following a further wash, 100uL of a sheep anti-complement C1q peroxidase conjugated antibody may beadded to each well and incubated for 1 hour at room temperature. Theplate may again be washed with wash buffer and 100 ul of substratebuffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) maybe added to each well. The oxidation reaction, observed by theappearance of a yellow color, may be allowed to proceed for 30 minutesand stopped by the addition of 100 ul of 4.5 NH2SO4. The absorbance maythen read at (492-405) nm.

A preferred variant in accordance with the invention is one thatdisplays a significant reduction in C1q binding, as detected andmeasured in this assay or a similar assay. Preferably the moleculecomprising an Fc variant displays about 50 fold reduction, about 60fold, about 80 fold, or about 90 fold reduction in C1q binding comparedto a control antibody having a nonmutated IgG1 Fc region. In the mostpreferred embodiment, the molecule comprising an Fc variant does notbind C1q, i.e. the variant displays about 100 fold or more reduction inC1q binding compared to the control antibody.

Another exemplary variant is one which has a better binding affinity forhuman C1q than the molecule comprising wild type Fc region. Such amolecule may display, for example, about two-fold or more, andpreferably about five-fold or more, improvement in human C1q bindingcompared to the parent molecule comprising wild type Fc region. Forexample, human C1q binding may be about two-fold to about 500-fold, andpreferably from about two-fold or from about five-fold to about1000-fold improved compared to the molecule comprising wild type Fcregion.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, e.g. as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996), which is incorporated herein byreference in its entirety. Briefly, various concentrations of themolecule comprising a variant Fc region and human complement may bediluted with buffer. Cells which express the antigen to which themolecule comprising a variant Fc region binds may be diluted to adensity of about 1×10⁶ cells/ml. Mixtures of the molecule comprising avariant Fc region, diluted human complement and cells expressing theantigen may be added to a flat bottom tissue culture 96 well plate andallowed to incubate for 2 hrs at 37 C and 5% CO₂ to facilitatecomplement mediated cell lysis. 50 μL of alamar blue (AccumedInternational) may then be added to each well and incubated overnight at37 C. The absorbance is measured using a 96-well fluorometer withexcitation at 530 nm and emission at 590 nm. The results may beexpressed in relative fluorescence units (RFU). The sampleconcentrations may be computed from a standard curve and the percentactivity as compared to nonvariant molecule, i.e., a molecule comprisingwild type Fc region, is reported for the variant of interest.

In some embodiments, an Fc variant of the invention does not activatecomplement. Preferably, the variant does not appear to have any CDCactivity in the above CDC assay. The invention also pertains to avariant with enhanced CDC compared to a parent molecule (a moleculecomprising wild type Fc region), e.g., displaying about two-fold toabout 100-fold improvement in CDC activity in vitro or in vivo (e.g., atthe IC50 values for each molecule being compared). Complement assays maybe performed with guinea pig, rabbit or human serum. Complement lysis oftarget cells may be detected by monitoring the release of intracellularenzymes such as lactate dehydrogenase (LDH), as described inKorzeniewski et al., 1983 Immunol. Methods 64(3): 313-20; and Decker etal., 1988 J. Immunol. Methods 115(1): 61-9, each of which isincorporated herein by reference in its entirety; or the release of anintracellular label such as europium, chromium 51 or indium 111 in whichtarget cells are labeled as described herein.

6.2.3 Other Assays

The molecules of the invention comprising variant Fc regions may also beassayed using any surface plasmon resonance based assays known in theart for characterizing the kinetic parameters of Fc-FcγR interactionbinding. Any SPR instrument commercially available including, but notlimited to, BIAcore Instruments, available from Biacore AB (Uppsala,Sweden); IAsys instruments available from Affinity Sensors (Franklin,Mass.); IBIS system available from Windsor Scientific Limited (Berks,UK), SPR-CELLIA systems available from Nippon Laser and Electronics Lab(Hokkaido, Japan), and SPR Detector Spreeta available from TexasInstruments (Dallas, Tex.) can be used in the instant invention. For areview of SPR-based technology see Mullet et al., 2000, Methods 22:77-91; Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash etal., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al.,2000, Current Opinion in Biotechnology 11: 54-61; all of which areincorporated herein by reference in their entirety. Additionally, any ofthe SPR instruments and SPR based methods for measuring protein-proteininteractions described in U.S. Pat. Nos. 6,373,577; 6,289,286;5,322,798; 5,341,215; 6,268,125 are contemplated in the methods of theinvention, all of which are incorporated herein by reference in theirentirety.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occurs is the sensor chip, which is at the heart ofthe SPR technology; it consists of a glass surface coated with a thinlayer of gold and forms the basis for a range of specialized surfacesdesigned to optimize the binding of a molecule to the surface. A varietyof sensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to, direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups, and attachmentthrough the histidine tag with NTA chips.

In some embodiments, the kinetic parameters of the binding of moleculesof the invention comprising variant Fc regions, e.g., immunoglobulinscomprising variant Fc region, to an FcγR may be determined using aBIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc.,Piscataway, N.J.). Any FcγR can be used to assess the interaction withthe molecules of the invention comprising variant Fc regions. In aspecific embodiment the FcγR is FcγRIIIA, preferably a soluble monomericFcγRIIIA. For example, in one embodiment, the soluble monomeric FcγRIIIAis the extracellular region of FcγRIIIA joined to the linker-AVITAGsequence (see, U.S. Provisional Application No. 60/439,498, filed onJan. 9, 2003 and U.S. Provisional Application No. 60/456,041 filed onMar. 19, 2003, which are incorporated herein by reference in theirentireties). In another specific embodiment, the FcγR is FcγRIIB,preferably a soluble dimeric FcγRIIB. For example in one embodiment, thesoluble dimeric FcγRIIB protein is prepared in accordance with themethodology described in U.S. Provisional application No. 60/439,709filed on Jan. 13, 2003, which is incorporated herein by reference in itsentirety.

An exemplary assay for determining the kinetic parameters of a moleculecomprising a variant Fc region, wherein the molecule is the 4-4-20antibody, to an FcγR using a BIAcore instrument comprises the following:BSA-FITC is immobilized on one of the four flow cells of a sensor chipsurface, preferably through amine coupling chemistry such that about5000 response units (RU) of BSA-FITC is immobilized on the surface. Oncea suitable surface is prepared, 4-4-20 antibodies carrying the Fcmutations are passed over the surface, preferably by one minuteinjections of a 20 μg/mL solution at a 5 μL/mL flow rate. The level of4-4-20 antibodies bound to the surface ranges between 400 and 700 RU.Next, dilution series of the receptor (FcγRIIA and FcγRIIB-Fc fusionprotein) in HBS-P buffer (20 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.5)are injected onto the surface at 100 μL/min. Antibody regenerationbetween different receptor dilutions is carried out preferably by single5 second injections of 100 mM NaHCO₃ pH 9.4; 3M NaCl. Any regenerationtechnique known in the art is contemplated in the method of theinvention.

Once an entire data set is collected, the resulting binding curves areglobally fitted using computer algorithms supplied by the SPR instrumentmanufacturer, e.g., BIAcore, Inc. (Piscataway, N.J.). These algorithmscalculate both the K_(on) and K_(off), from which the apparentequilibrium binding constant, K_(d) is deduced as the ratio of the tworate constants (i.e., K_(off)/K_(on)). More detailed treatments of howthe individual rate constants are derived can be found in theBIAevaluation Software Handbook (BIAcore, Inc., Piscataway, N.J.). Theanalysis of the generated data may be done using any method known in theart. For a review of the various methods of interpretation of thekinetic data generated see Myszka, 1997, Current Opinion inBiotechnology 8: 50-7; Fisher et al., 1994, Current Opinion inBiotechnology 5: 389-95; O'Shannessy, 1994, Current Opinion inBiotechnology, 5:65-71; Chaiken et al., 1992, Analytical Biochemistry,201: 197-210; Morton et al., 1995, Analytical Biochemistry 227: 176-85;O'Shannessy et al., 1996, Analytical Biochemistry 236: 275-83; all ofwhich are incorporated herein by reference in their entirety.

In preferred embodiments, the kinetic parameters determined using an SPRanalysis, e.g., BIAcore, may be used as a predictive measure of how amolecule of the invention will function in a functional assay, e.g.,ADCC. An exemplary method for predicting the efficacy of a molecule ofthe invention based on kinetic parameters obtained from an SPR analysismay comprise the following: determining the K_(off) values for bindingof a molecule of the invention to FcγRIIIA and FcγRIIB; plotting (1)K_(off) (wt)/K_(off) (mut) for FcγRIIIA; (2) K_(off) (mut)/K_(off) (wt)for FcγRIIB against the ADCC data. Numbers higher than one show adecreased dissociation rate for FcγRIIIA and an increased dissociationrate for FcγRIIB relative to wild type; and possess and enhanced ADCCfunction.

6.3 Methods of Recombinantly Producing Molecules of the Invention

6.3.1 Polynucleotides Encoding Molecules of the Invention

The present invention also includes polynucleotides that encode themolecules, including the polypeptides and antibodies, of the inventionidentified by the methods of the invention. The polynucleotides encodingthe molecules of the invention may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart.

Once the nucleotide sequence of the molecules (e.g., antibodies) thatare identified by the methods of the invention is determined, thenucleotide sequence may be manipulated using methods well known in theart, e.g., recombinant DNA techniques, site directed mutagenesis, PCR,etc. (see, for example, the techniques described in Sambrook et al.,2001, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.; and Ausubel et al., eds.,1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY,which are both incorporated by reference herein in their entireties), togenerate, for example, antibodies having a different amino acidsequence, for example by generating amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, when the nucleic acids encode antibodies, oneor more of the CDRs are inserted within framework regions using routinerecombinant DNA techniques. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 fora listing of human framework regions).

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the molecules of the invention.

6.3.2 Recombinant Expression of Molecules of the Invention

Once a nucleic acid sequence encoding molecules of the invention (i.e.,antibodies) has been obtained, the vector for the production of themolecules may be produced by recombinant DNA technology using techniqueswell known in the art. Methods which are well known to those skilled inthe art can be used to construct expression vectors containing thecoding sequences for the molecules of the invention and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, for example, thetechniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of a moleculeidentified by the methods of the invention (i.e., an antibody) can betransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the molecules of the invention. Inspecific embodiments, the expression of the molecules of the inventionis regulated by a constitutive, an inducible or a tissue, specificpromoter.

The host cells used to express the molecules identified by the methodsof the invention may be either bacterial cells such as Escherichia coli,or, preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al., 1998, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to expressthe molecules identified by the methods of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof the molecules of the invention may be produced and subsequentlypurified, but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, expressthe molecules of the invention in situ. These include, but are notlimited to, microorganisms such as bacteria (e.g., E. coli and B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing coding sequences for themolecules identified by the methods of the invention; yeast (e.g.,Saccharomyces Pichia) transformed with recombinant yeast expressionvectors containing sequences encoding the molecules identified by themethods of the invention; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing the sequencesencoding the molecules identified by the methods of the invention; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing sequences encoding the molecules identified by themethods of the invention; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (human retinal cells developed by Crucell)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the moleculebeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibodycoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free gluta-thione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977. Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly knownin the art of recombinant DNA technology which can be used are describedin Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, JohnWiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1;and hygro, which confers resistance to hygromycin (Santerre et al.,1984, Gene 30:147).

The expression levels of an antibody of the invention can be increasedby vector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987). When a marker in the vector system expressing an antibodyis amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (Crouse et al.,1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once a molecule of the invention (i.e., antibodies) has beenrecombinantly expressed, it may be purified by any method known in theart for purification of polypeptides or antibodies, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of polypeptides orantibodies.

6.4 Prophylactic and Therapeutic Methods

The present invention encompasses administering one or more of themolecules of the invention (e.g., antibodies) to an animal, preferably amammal, and most preferably a human, for preventing, treating, orameliorating one or more symptoms associated with a disease, disorder,or infection. The molecules of the invention are particularly useful forthe treatment or prevention of a disease or disorder where an enhancedefficacy of effector cell function (e.g., ADCC) mediated by FcγR isdesired. The methods and compositions of the invention are particularlyuseful for the treatment or prevention of primary or metastaticneoplastic disease (i.e., cancer), and infectious diseases. Molecules ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein. As detailedbelow, the molecules of the invention can be used in methods of treatingor preventing cancer (particularly in passive immunotherapy), autoimmunedisease, inflammatory disorders or infectious diseases.

The molecules of the invention may also be advantageously utilized incombination with other therapeutic agents known in the art for thetreatment or prevention of a cancer, autoimmune disease, inflammatorydisorders or infectious diseases. In a specific embodiment, molecules ofthe invention may be used in combination with monoclonal or chimericantibodies, lymphokines, or hematopoietic growth factors (such as, e.g.,IL-2, IL-3 and IL-7), which, for example, serve to increase the numberor activity of effector cells which interact with the molecules and,increase immune response. The molecules of the invention may also beadvantageously utilized in combination with one or more drugs used totreat a disease, disorder, or infection such as, for example anti-canceragents, anti-inflammatory agents or anti-viral agents, e.g., as detailedin Sections 6.4.1.2 and 6.4.2.1 below.

6.4.1 Cancers

The invention encompasses methods and composition for treatment orprevention of cancer or metastasis in a subject comprising administeringto the subject a therapeutically effective amount of one or moremolecules comprising a variant Fc region.

Molecules of the invention (i.e., polypeptides, antibodies) comprisingvariant Fc regions can be used to prevent, inhibit or reduce the growthof primary tumors or metastasis of cancerous cells. In one embodiment,the molecule of the invention comprises a variant Fc that binds FcγRIIIAand/or FcγRIIA with a greater affinity than a comparable polypeptidecomprising a wild type Fc region binds FcγRIIIA and/or FcγRIIA, and/orsaid variant Fc region has an enhanced effector function, e.g., ADCC,CDC, phagocytosis, opsonization, etc. Such molecules can be used aloneto treat or prevent cancer. In another embodiment, the molecule of theinvention comprises a variant Fc region that binds FcγRIIIA and/orFcγRIIA with a greater affinity than a comparable polypeptide comprisinga wild type Fc region binds FcγRIIIA and/or FcγRIIA, and further bindsFcγRIIB with a lower affinity than a comparable polypeptide comprising awild-type Fc region binds FcγRIIB, and/or said variant Fc region has anenhanced effector function, e.g., ADCC, CDC, phagocytosis, opsonization,etc. Such molecules can also be used alone to treat or prevent cancer.

In some embodiments, the invention encompasses methods and compositionsfor the treatment or prevention of cancer in a subject with FcγRpolymorphisms such as those homozygous for the FγRIIIA-158V orFcγRIIIA-158F alleles. In some embodiments, the invention encompassesengineering therapeutic antibodies, e.g., tumor specific monoclonalantibodies in accordance with the methods of the invention such that theengineered antibodies have enhanced efficacy in patients homozygous forthe low affinity allele of FcγRIIIA (158F). In other embodiments, theinvention encompasses engineering therapeutic antibodies, e.g., tumorspecific monoclonal antibodies in accordance with the methods of theinvention such that the engineered antibodies have enhanced efficacy inpatients homozygous for the high affinity allele of FcγRIIIA (158V).

In some embodiments, the engineered antibodies of the invention areparticularly effective in treating and/or preventing non-Hodgkin'slymphoma (NHL). The engineered antibodies of the invention aretherapeutically more effective than current therapeutic regimens forNHL, including but not limited to chemotherapy, and immunotherapy usinganti-CD20 mAb, Rituximab. The efficacy of anti-CD20 monoclonalantibodies however depends on the FcγR polymorphism of the subject(Carton et al., 2002 Blood, 99: 754-8; Weng et al., 2003 J Clin Oncol.21(21):3940-7 both of which are incorporated herein by reference intheir entireties). These receptors are expressed on the surface of theeffector cells and mediate ADCC. High affinity alleles, of the lowaffinity activating receptors, improve the effector cells' ability tomediate ADCC. The methods of the invention allow engineering anti-CD20antibodies harboring Fc mutations to enhance their affinity to FcγR oneffector cells via their altered Fc domains. The engineered antibodiesof the invention provide better immunotherapy reagents for patientsregardless of their FcγR polymorphism.

An exemplary method for determining the efficacy of the engineeredanti-CD20 antibodies in a subject may include the following: Plasmidsharboring chimeric anti-HER2/neu heavy chain genes with Fc mutationsthat show substantially increased killing in ADCC can be used as abackbone to transfer in the variable domain from the Rituximab heavychain gene. The variable region from the anti-HER2/neu Fc variant isreplaced with the variable region from Rituximab. Plasmids containingwild type Fc domains or a D265A mutation to abrogate FcR binding, or theanti-CD20 Fc variants are transiently cotransfected with the Rituximablight chain gene into 293H cells, conditioned media and the antibody ispurified over a protein G column using routine methods.

Anti-CD20 mAbs harboring the Fc variants are tested by ADCC using acultured B cell line to determine the ability of the Fc mutations toenhance ADCC. Standard ADCC is performed using methods disclosed herein.Lymphocytes are harvested from peripheral blood using a Ficoll-Paquegradient (Pharmacia). Target Daudi cells, a B-cell line expressing CD20,are loaded with Europium (PerkinElmer) and incubated with effectors for4 hrs at 37° C. Released Europium is detected using a fluorescent platereader (Wallac). The resulting ADCC data indicates the efficacy of theFc variants to trigger NK cell mediated cytotoxicity and establish whichanti-CD20 Fc variants can be tested with both patient samples andelutriated monocytes. Fc variants showing the greatest potential forenhancing the efficacy of the anti-CD20 antibody are then tested in anADCC assay using PBMCs from patients. PBMC from healthy donors are usedas effector cells. In vitro ADCC assays using anti-CD20 variants andRituximab are performed in primary lymphoma cells from patients withfollicular lymphoma. The specific FcγR polymorphism of the donors isdetermined and cataloged using methods known in the art. ADCC assay isperformed by effector cells from patients with different FcγRIIIA andFcγRIIA genotypes.

According to an aspect of the invention, molecules (e.g., antibodies) ofthe invention comprising variant Fc regions enhance the efficacy ofcancer immunotherapy by increasing the potency of the antibody effectorfunction relative to a molecule containing the wild-type Fc region,e.g., ADCC, CDC, phagocytosis, opsonization, etc. In a specificembodiment, antibody dependent cellular toxicity and/or phagocytosis oftumor cells is enhanced using the molecules of the invention withvariant Fc regions. Molecules of the invention may enhance the efficacyof immunotherapy cancer treatment by enhancing at least oneantibody-mediated effector function. In one particular embodiment, amolecule of the invention comprising a variant Fc region enhances theefficacy of immunotherapy treatment by enhancing the complementdependent cascade. In another embodiment of the invention, the moleculeof the invention comprising a variant Fc region enhances the efficacy ofimmunotherapy treatment by enhancing the phagocytosis and/oropsonization of the targeted tumor cells. In another embodiment of theinvention, the molecule of the invention comprising a variant Fc regionenhances the efficacy of treatment by enhancing antibody-dependentcell-mediated cytotoxicity (“ADCC”) in destruction of the targeted tumorcells.

The invention further contemplates engineering therapeutic antibodies(e.g., tumor specific monoclonal antibodies) for enhancing thetherapeutic efficacy of the therapeutic antibody, for example, byenhancing the effector function of the therapeutic antibody (e.g.,ADCC). Preferably the therapeutic antibody is a cytotoxic and/oropsonizing antibody. It will be appreciated by one of skill in the art,that once molecules of the invention with desired binding properties(e.g., molecules with variant Fc regions with at least one amino acidmodification, which modification enhances the affinity of the variant Fcregion for FcγRIIIA and/or FcγRIIA relative to a comparable molecule,comprising a wild-type Fc region) have been identified (See Section 6.2and Table 9) according to the methods of the invention, therapeuticantibodies may be engineered using standard recombinant DNA techniquesand any known mutagenesis techniques, as described in Section 6.2.2 toproduce engineered therapeutic carrying the identified mutation siteswith the desired binding properties. Any of the therapeutic antibodieslisted in Table 10 that have demonstrated therapeutic utility in cancertreatment, may be engineered according to the methods of the invention,for example, by modifying the Fc region to have an enhanced affinity forFcγRIIIA and/or FcγRIIA compared to a therapeutic antibody having awild-type Fc region, and used for the treatment and or prevention of acancer characterized by a cancer antigen. Other therapeutic antibodiesinclude those against pathogenic agents such as those againstStreptococcus pneumoniae Serotype 6B, see, e.g., Sun et al., 1999,Infection and Immunity, 67(3): 1172-9.

The Fc variants of the invention may be incorporated into therapeuticantibodies such as those disclosed herein or other Fc fusion clinicalcandidates, i.e., a molecule comprising an Fc regions which has beenapproved for us in clinical trials or any other molecule that maybenefit from the Fc variants of the instant invention, humanized,affinity matured, modified or engineered versions thereof.

The invention also encompasses engineering any other polypeptidecomprising an Fc region which has therapeutic utility, including but notlimited to ENBREL, according to the methods of the invention, in orderto enhance the therapeutic efficacy of such polypeptides, for example,by enhancing the effector function of the polypeptide comprising an Fcregion.

TABLE 10 THERAPEUTIC ANTIBODIES THAT CAN BE ENGINEERED ACCORDING TO THEMETHODS OF THE INVENTION Company Product Disease Target AbgenixABX-EGF ™ Cancer EGF receptor AltaRex OvaRex ™ ovarian cancer tumorantigen CA125 BravaRex ™ metastatic cancers tumor antigen MUC1 AntisomaTheragyn ™ ovarian cancer PEM antigen (pemtumomabytrrium-90) Therex ™breast cancer PEM antigen Boehringer Blvatuzumab head & neck cancer CD44Ingelheim Centocor/J&J Panorex ™ Colorectal cancer 17-1A ReoPro ™ PTCAgp IIIb/IIIa ReoPro ™ Acute MI gp IIIb/IIIa ReoPro ™ Ischemic stroke gpIIIb/IIIa Corixa Bexocar ™ NHL CD20 CRC Technology MAb, idiotypic 105AD7colorectal cancer gp72 vaccine Crucell Anti-EpCAM cancer Ep-CAMCytoclonal MAb, lung cancer non-small cell lung NA cancer GenentechHerceptin ® metastatic breast HER-2 cancer Herceptin ® early stagebreast HER-2 cancer Rituxan ® Relapsed/refractory CD20 low-grade orfollicular NHL Rituxan ® intermediate & CD20 high-grade NHL MAb-VEGFNSCLC, metastatic VEGF MAb-VEGF Colorectal cancer, VEGF metastatic AMD ™Fab age-related macular CD18 degeneration E-26 ™ (2^(nd) gen. IgE)allergic asthma & IgE rhinitis IDEC Zevalin ™ (Rituxan ™ + low grade ofCD20 yttrium-90) follicular, relapsed or refractory, CD20- positive,B-cell NHL and Rituximab- refractory NHL ImClone Cetuximab ™ + innotecanrefractory colorectal EGF receptor carcinoma Cetuximab ™ + cisplatin &newly diagnosed or EGF receptor radiation recurrent head & neck cancerCetuximab ™ + gemcitabine newly diagnosed EGF receptor metastaticpancreatic carcinoma Cetuximab ™ + cisplatin + recurrent or EGF receptor5FU or Taxol metastatic head & neck cancer Cetuximab ™ + carboplatin +newly diagnosed EGF receptor paclitaxel non-small cell lung carcinomaCetuximab ™ + cisplatin ™ head & neck cancer EGF receptor (extensiveincurable local-regional disease & distant metasteses) Cetuximab +radiation locally advanced EGF receptor head & neck carcinoma BEC2 +Bacillus Calmette small cell lung mimics ganglioside GD3 Guerincarcinoma BEC2 + Bacillus Calmette melanoma mimics ganglioside GD3Guerin IMC-1C11 colorectal cancer VEGF-receptor with liver metastesesImmonoGen nuC242-DM1 Colorectal, gastric, nuC242 and pancreatic cancerImmunoMedics LymphoCide ™ Non-Hodgkins CD22 lymphoma LymphoCide Y-90 ™Non-Hodgkins CD22 lymphoma CEA-Cide ™ metastatic solid CEA tumorsCEA-Cide Y-90 ™ metastatic solid CEA tumors CEA-Scan ™ (Tc-99m-colorectal cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan ™(Tc-99m- Breast cancer CEA labeled arcitumomab) (radioimaging)CEA-Scan ™ (Tc-99m- lung cancer CEA labeled arcitumomab) (radioimaging)CEA-Scan ™ (Tc-99m- intraoperative CEA labeled arcitumomab) tumors(radio imaging) LeukoScan ™ (Tc-99m- soft tissue infection CEA labeledsulesomab) (radioimaging) LymphoScan ™ (Tc-99m- lymphomas CD22 labeled)(radioimaging) AFP-Scan ™ (Tc-99m- liver 7 gem-cell AFP labeled) cancers(radioimaging) Intracel HumaRAD-HN (+ yttrium- head & neck cancer NA 90)HumaSPECT colorectal imaging NA Medarex MDX-101 (CTLA-4) Prostate andother CTLA-4 cancers MDX-210 (her-2 Prostate cancer HER-2overexpression) MDX-210/MAK Cancer HER-2 MedImmune Vitaxin ™ Cancer αvβ₃Merck KGaA MAb 425 Various cancers EGF receptor IS-IL-2 Various cancersEp-CAM Millennium Campath ® (alemtuzumab) chronic lymphocytic CD52leukemia NeoRx CD20-streptavidin (+ biotin- Non-Hodgkins CD20 yttrium90) lymphoma Avidicin (albumin + metastatic cancer NA NRLU13) PeregrineOncolym ™ (+ iodine-131) Non-Hodgkins HLA-DR 10 beta lymphoma Cotara ™(+ iodine-131) unresectable DNA-associated proteins malignant gliomaPharmacia C215 (+ staphylococcal pancreatic cancer NA Corporationenterotoxin) MAb, lung/kidney cancer lung & kidney NA cancer nacolomabtafenatox (C242 + colon & pancreatic NA staphylococcal cancerenterotoxin) Protein Design Nuvion ™ T cell malignancies CD3 Labs SMARTM195 ™ AML CD33 SMART 1D10 ™ NHL HLA-DR antigen Titan CEAVac ™colorectal cancer, CEA advanced TriGem ™ metastatic GD2-gangliosidemelanoma & small cell lung cancer TriAb ™ metastatic breast MUC-1 cancerTrilex CEAVa ™ c colorectal cancer, CEA advanced TriGem ™ metastaticGD2-ganglioside melanoma & small cell lung cancer TriAb ™ metastaticbreast MUC-1 cancer Viventia Biotech NovoMAb-G2 radiolabeledNon-Hodgkins NA lymphoma Monopharm C ™ colorectal & SK-1 antigenpancreatic carcinoma GlioMAb-H ™ (+ gelonin gliorna, melanoma NA toxin)& neuroblastoma Xoma Rituxan ™ Relapsed/refractory CD20 low-grade orfollicular NHL Rituxan ™ intermediate & CD20 high-grade NHL ING-1adenomcarcinoma Ep-CAM

Accordingly, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen, using a therapeutic antibodythat binds a cancer antigen and is cytotoxic and has been modified atone or more sites in the Fc region, according to the invention, to bindFcγRIIIA and/or FcγRIIA with a higher affinity than the parenttherapeutic antibody, and/or mediates effector function (e.g., ADCC,phagocytosis) more effectively. In another embodiment, the inventionprovides methods of preventing or treating cancer characterized by acancer antigen, using a therapeutic antibody that binds a cancer antigenand is cytotoxic, and has been engineered according to the invention tobind FcγRIIIA and/or FcγRIIA with a higher affinity and bind FcγRIIBwith a lower affinity than the parent therapeutic antibody, and/ormediates effector function (e.g., ADCC, phagocytosis) more effectively.The therapeutic antibodies that have been engineered according to theinvention are useful for prevention or treatment of cancer, since theyhave an enhanced cytotoxic activity (e.g., enhanced tumor cell killingand/or enhanced for example, ADCC activity or CDC activity).

Cancers associated with a cancer antigen may be treated or prevented byadministration of a therapeutic antibody that binds a cancer antigen andis cytotoxic, and has been engineered according to the methods of theinvention to have, for example, an enhanced effector function. In oneparticular embodiment, the therapeutic antibodies engineered accordingto the methods of the invention enhance the antibody-mediated cytotoxiceffect of the antibody directed at the particular cancer antigen. Forexample, but not by way of limitation, cancers associated with thefollowing cancer antigens may be treated or prevented by the methods andcompositions of the invention: KS ¼ pan-carcinoma antigen (Perez andWalker, 1990, J. Immunol. 142:32-37; Bumal, 1988, Hybridoma7(4):407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991,Cancer Res. 51(2):48-475), prostatic acid phosphate (Tailor et al.,1990, Nucl. Acids Res. 18(1):4928), prostate specific antigen (Henttuand Vihko, 1989, Biochem. Biophys. Res. Comm. 10(2):903-910; Israeli etal., 1993, Cancer Res. 53:227-230), melanoma-associated antigen p97(Estin et al., 1989, J. Natl. Cancer Instit. 81(6):445-44), melanomaantigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med.171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA)(Natali et al., 1987, Cancer 59:55-3; Mittelman et al., 1990, J. Clin.Invest. 86:2136-2144)), prostate specific membrane antigen,carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin.Oncol. 13:294), polymorphic epithelial mucin antigen, human milk fatglobule antigen, Colorectal tumor-associated antigens such as: CEA,TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO17-1A(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlynet al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336),human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445),CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specificantigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151,3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J.Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, CancerRes. 53:5244-5250), tumor-specific transplantation type of cell-surfaceantigen (TSTA) such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses,oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403), neoglycoprotein,sphingolipids, breast cancer antigen such as EGFR (Epidermal growthfactor receptor), HER2 antigen (p185^(HER2)), polymorphic epithelialmucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359),malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science245:301-304), differentiation antigen (Feizi, 1985, Nature 314:53-57)such as I antigen found in fetal erythrocytes and primary endoderm,I(Ma) found in gastric adenocarcinomas, M18 and M39 found in breastepithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, andD₁56-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 foundin colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found ingastric cancer. Y hapten, Le^(y) found in embryonal carcinoma cells, TL5(blood group A), EGF receptor found in A431 cells, E₁ series (bloodgroup B) found in pancreatic cancer, FC10.2 found in embryonal carcinomacells, gastric adenocarcinoma, CO-514 (blood group Le^(a)) found inadenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood groupLe^(a)), G49, EGF receptor, (blood group ALe^(b)/Le^(y)) found incolonic adenocarcinoma, 19.9 found in colon cancer, gastric cancermucins, T₅A₇ found in myeloid cells, R₂₄ found in melanoma, 4.2, G_(D3),D1.1, OFA-1, G_(M2), OFA-2, G_(D2), M1:22:25:8 found in embryonalcarcinoma cells and SSEA-3, SSEA-4 found in 4-8-cell stage embryos. Inanother embodiment, the antigen is a T cell receptor derived peptidefrom a cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal4:62).

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: Leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, papillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including but not limitedto, squamous cell cancer, and verrucous; skin cancers including but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter);Wilms' tumor; bladder cancers including but not limited to, transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroidand skin; including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; othertumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

In a specific embodiment, a molecule of the invention (e.g., an antibodycomprising a variant Fc region, or a therapeutic monoclonal antibodyengineered according to the methods of the invention) inhibits orreduces the growth of primary tumor or metastasis of cancerous cells byat least 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth of primary tumor ormetastasis in the absence of said molecule of the invention.

6.4.1.1 Combination Therapy

The invention further encompasses administering the molecules of theinvention in combination with other therapies known to those skilled inthe art for the treatment or prevention of cancer, including but notlimited to, current standard and experimental chemotherapies, hormonaltherapies, biological therapies, immunotherapies, radiation therapies,or surgery. In some embodiments, the molecules of the invention may beadministered in combination with a therapeutically or prophylacticallyeffective amount of one or more anti-cancer agents, therapeuticantibodies (e.g., antibodies listed in Table 10), or other agents knownto those skilled in the art for the treatment and/or prevention ofcancer (See Section 6.4.1.2).

In certain embodiments, one or more molecule of the invention isadministered to a mammal, preferably a human, concurrently with one ormore other therapeutic agents useful for the treatment of cancer. Theterm “concurrently” is not limited to the administration of prophylacticor therapeutic agents at exactly the same time, but rather it is meantthat a molecule of the invention and the other agent are administered toa mammal in a sequence and within a time interval such that the moleculeof the invention can act together with the other agent to provide anincreased benefit than if they were administered otherwise. For example,each prophylactic or therapeutic agent (e.g., chemotherapy, radiationtherapy, hormonal therapy or biological therapy) may be administered atthe same time or sequentially in any order at different points in time;however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute. In various embodiments, the prophylactic or therapeutic agentsare administered less than 1 hour apart, at about 1 hour apart, at about1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart,at about 3 hours to about 4 hours apart, at about 4 hours to about 5hours apart, at about 5 hours to about 6 hours apart, at about 6 hoursto about 7 hours apart, at about 7 hours to about 8 hours apart, atabout 8 hours to about 9 hours apart, at about 9 hours to about 10 hoursapart, at about 10 hours to about 11 hours apart, at about 11 hours toabout 12 hours apart, no more than 24 hours apart or no more than 48hours apart. In preferred embodiments, two or more components areadministered within the same patient visit.

In other embodiments, the prophylactic or therapeutic agents areadministered at about 2 to 4 days apart, at about 4 to 6 days apart, atabout 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart. In preferred embodiments, the prophylactic or therapeutic agentsare administered in a time frame where both agents are still active. Oneskilled in the art would be able to determine such a time frame bydetermining the half life of the administered agents.

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a subject. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of a second agent and/or third agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

In certain embodiments, prophylactic or therapeutic agents areadministered in a cycle of less than about 3 weeks, about once every twoweeks, about once every 10 days or about once every week. One cycle cancomprise the administration of a therapeutic or prophylactic agent byinfusion over about 90 minutes every cycle, about 1 hour every cycle,about 45 minutes every cycle. Each cycle can comprise at least 1 week ofrest, at least 2 weeks of rest, at least 3 weeks of rest. The number ofcycles administered is from about 1 to about 12 cycles, more typicallyfrom about 2 to about 10 cycles, and more typically from about 2 toabout 8 cycles.

In yet other embodiments, the therapeutic and prophylactic agents of theinvention are administered in metronomic dosing regimens, either bycontinuous infusion or frequent administration without extended restperiods. Such metronomic administration can involve dosing at constantintervals without rest periods. Typically the therapeutic agents, inparticular cytotoxic agents, are used at lower doses. Such dosingregimens encompass the chronic daily administration of relatively lowdoses for extended periods of time. In preferred embodiments, the use oflower doses can minimize toxic side effects and eliminate rest periods.In certain embodiments, the therapeutic and prophylactic agents aredelivered by chronic low-dose or continuous infusion ranging from about24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3weeks to about 1 month to about 2 months, to about 3 months, to about 4months, to about 5 months, to about 6 months. The scheduling of suchdose regimens can be optimized by the skilled oncologist.

In other embodiments, courses of treatment are administered concurrentlyto a mammal, i.e., individual doses of the therapeutics are administeredseparately yet within a time interval such that molecules of theinvention can work together with the other agent or agents. For example,one component may be administered one time per week in combination withthe other components that may be administered one time every two weeksor one time every three weeks. In other words, the dosing regimens forthe therapeutics are carried out concurrently even if the therapeuticsare not administered simultaneously or within the same patient visit.

When used in combination with other prophylactic and/or therapeuticagents, the molecules of the invention and the prophylactic and/ortherapeutic agent can act additively or, more preferably,synergistically. In one embodiment, a molecule of the invention isadministered concurrently with one or more therapeutic agents in thesame pharmaceutical composition. In another embodiment, a molecule ofthe invention is administered concurrently with one or more othertherapeutic agents in separate pharmaceutical compositions. In stillanother embodiment, a molecule of the invention is administered prior toor subsequent to administration of another prophylactic or therapeuticagent. The invention contemplates administration of a molecule of theinvention in combination with other prophylactic or therapeutic agentsby the same or different routes of administration, e.g., oral andparenteral. In certain embodiments, when a molecule of the invention isadministered concurrently with another prophylactic or therapeutic agentthat potentially produces adverse side effects including, but notlimited to, toxicity, the prophylactic or therapeutic agent canadvantageously be administered at a dose that falls below the thresholdthat the adverse side effect is elicited.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

6.4.1.2 Other Therapeutic/Prophylactic Agents

In a specific embodiment, the methods of the invention encompass theadministration of one or more molecules of the invention with one ormore therapeutic agents used for the treatment and/or prevention ofcancer. In one embodiment, angiogenesis inhibitors may be administeredin combination with the molecules of the invention. Angiogenesisinhibitors that can be used in the methods and compositions of theinvention include but are not limited to: Angiostatin (plasminogenfragment); antiangiogenic antithrombin III; Angiozyme; ABT-627; Bay12-9566; Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor(CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; CombretastatinA-4; Endostatin (collagen XVIII fragment); Fibronectin fragment;Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment;HMV833; Human chorionic gonadotropin (hCG); IM-862; Interferonalpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin-12;Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinaseinhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAbIMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placental ribonucleaseinhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4);Prinomastat; Prolactin 16 kD fragment; Proliferin-related protein (PRP);PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416;SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide;Thrombospondin-1 (TSP-1); TNP-470; Transforming growth factor-beta(TGF-b); Vasculostatin; Vasostatin (caireticulin fragment); ZD6126; ZD6474; farnesyl transferase inhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with the molecules ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to ZENAPAX® (daclizumab) (RochePharmaceuticals, Switzerland) which is an immunosuppressive, humanizedanti-CD25 monoclonal antibody for the prevention of acute renalallograft rejection; PANOREX™ which is a murine anti-17-IA cell surfaceantigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 is a human anti-TGF-β₂ antibody (Cambridge Ab Tech).Other examples of therapeutic antibodies that can be used in accordancewith the invention are presented in Table 10.

6.4.2 Autoimmune Disease and Inflammatory Diseases

In some embodiments, molecules of the invention comprise a variant Fcregion, having one or more amino acid modifications in one or moreregions, which modification increases the affinity of the variant Fcregion for FcγRIIB but decreases the affinity of the variant Fc regionfor FcγRIIIA and/or FcγRIIA. Molecules of the invention with suchbinding characteristics are useful in regulating the immune response,e.g., in inhibiting the immune response in connection with autoimmunediseases or inflammatory diseases. Although not intending to be bound byany mechanism of action, molecules of the invention with an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA and/orFcγRIIA may lead to dampening of the activating response to FcγR andinhibition of cellular responsiveness.

In some embodiments, a molecule of the invention comprising a variant Fcregion is not an immunoglobulin, and comprises at least one amino acidmodification which modification increases the affinity of the variant Fcregion for FcγRIIB relative to a molecule comprising a wild-type Fcregion. In other embodiments, said molecule further comprises one ormore amino acid modifications, which modifications decreases theaffinity of the molecule for an activating FcγR. In some embodiments,the molecule is a soluble (i.e., not membrane bound) Fc region. Theinvention contemplates other amino acid modifications within the solubleFc region which modulate its affinity for various Fc receptors,including those known to one skilled in the art as described herein. Inother embodiments, the molecule (e.g., the Fc region comprising at leastone or more amino acid modification) is modified using techniques knownto one skilled in the art and as described herein to increase the invivo half life of the Fc region. Such molecules have therapeutic utilityin treating and/or preventing an autoimmune disorder. Although notintending to be bound by any mechanism of actions, such molecules withenhanced affinity for FcγRIIB will lead to a dampening of the activatingreceptors and thus a dampening of the immune response and havetherapeutic efficacy for treating and/or preventing an autoimmunedisorder.

In certain embodiments, the one or more amino acid modifications, whichincrease the affinity of the variant Fc region for FcγRIIB but decreasethe affinity of the variant Fc region for FcγRIIIA comprise asubstitution at position 246 with threonine and at position 396 withhistidine; or a substitution at position 268 with aspartic acid and atposition 318 with aspartic acid; or a substitution at position 217 withserine, at position 378 with valine, and at position 408 with arginine;or a substitution at position 375 with cysteine and at position 396 withleucine; or a substitution at position 246 with isoleucine and atposition 334 with asparagine. In one embodiment, the one or more aminoacid modifications, which increase the affinity of the variant Fc regionfor FcγRIIB but decrease the affinity of the variant Fc region forFcγRIIIA comprise a substitution at position 247 with leucine. Inanother embodiment, the one or more amino acid modification, whichincreases the affinity of the variant Fc region for FcγRIIB butdecreases the affinity of the variant Fc region for FcγRIIIA comprise asubstitution at position 372 with tyrosine. In yet another embodiment,the one or more amino acid modification, which increases the affinity ofthe variant Fc region for FcγRIIB but decreases the affinity of thevariant Fc region for FcγRIIIA comprise a substitution at position 326with glutamic acid. In one embodiment, the one or more amino acidmodification, which increases the affinity of the variant Fc region forFcγRIIB but decreases the affinity of the variant Fc region for FcγRIIIAcomprise a substitution at position 224 with leucine.

The variant Fc regions that have an enhanced affinity for FcγRIIB and adecreased affinity for FcγRIIIA and/or FcγRIIA relative to a comparablemolecule comprising a wild-type Fc region, may be used to treat orprevent autoimmune diseases or inflammatory diseases. The presentinvention provides methods of preventing, treating, or managing one ormore symptoms associated with an autoimmune or inflammatory disorder ina subject, comprising administering to said subject a therapeutically orprophylactically effective amount of one or more molecules of theinvention with variant Fc regions that have an enhanced affinity forFcγRIIB and a decreased affinity for FcγRIIIA and or FcγRIIA relative toa comparable molecule comprising a wild type Fc region.

The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an inflammatory disorderin a subject further comprising, administering to said subject atherapeutically or prophylactically effective amount of one or moreanti-inflammatory agents. The invention also provides methods forpreventing, treating, or managing one or more symptoms associated withan autoimmune disease further comprising, administering to said subjecta therapeutically or prophylactically effective amount of one or moreimmunomodulatory agents. Section 6.4.2.1 provides non-limiting examplesof anti-inflammatory agents and immunomodulatory agents.

Examples of autoimmune disorders that may be treated by administeringthe molecules of the present invention include, but are not limited to,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Ménière's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteritis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein inSection 3.2.2, some autoimmune disorders are associated with aninflammatory condition. Thus, there is overlap between what isconsidered an autoimmune disorder and an inflammatory disorder.Therefore, some autoimmune disorders may also be characterized asinflammatory disorders. Examples of inflammatory disorders which can beprevented, treated or managed in accordance with the methods of theinvention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

Molecules of the invention with variant Fc regions that have an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild-type Fc region can also be used toreduce the inflammation experienced by animals, particularly mammals,with inflammatory disorders. In a specific embodiment, a molecule of theinvention reduces the inflammation in an animal by at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, at least 50%, at least 45%, at least 40%, atleast 45%, at least 35%, at least 30%, at least 25%, at least 20%, or atleast 10% relative to the inflammation in an animal, which is notadministered the said molecule.

Molecules of the invention with variant Fc regions that have an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild-type Fc region can also be used toprevent the rejection of transplants.

The invention further contemplates engineering any of the antibodiesknown in the art for the treatment and/or prevention of autoimmunedisease or inflammatory disease, so that the antibodies comprise avariant Fc region comprising one or more amino acid modifications, whichhave been identified by the methods of the invention to have an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild type Fc region. A non-limitingexample of the antibodies that are used for the treatment or preventionof inflammatory disorders which can be engineered according to theinvention is presented in Table 11, and a non-limiting example of theantibodies that are used for the treatment or prevention of autoimmunedisorder is presented in Table 12.

TABLE 11 ANTIBODIES FOR INFLAMMATORY DISEASES AND AUTOIMMUNE DISEASESTHAT CAN ENGINEERED IN ACCORDANCE WITH THE INVENTION. Antibody TargetProduct Name Antigen Type Isotype Sponsors Indication 5G1.1 ComplementHumanized IgG Alexion Pharm Rheumatoid Arthritis (C5) Inc 5G1.1Complement Humanized IgG Alexion Pharm SLE (C5) Inc 5G1.1 ComplementHumanized IgG Alexion Pharm Nephritis (C5) Inc 5G1.1-SC ComplementHumanized ScFv Alexion Pharm Cardiopulmonary (C5) Inc Bypass 5G1.1-SCComplement Humanized ScFv Alexion Pharm Myocardial Infarction (C5) Inc5G1.1-SC Complement Humanized ScFv Alexion Pharm Angioplasty (C5) IncABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgG Abgenix IncAllograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix Inc PsoriasisAntegren VLA-4 Humanized IgG Athena/Elan Multiple Sclerosis Anti-CD11aCD11a Humanized IgG1 Genentech Psoriasis Inc/Xoma Anti-CD18 CD18Humanized Fab′2 Genentech Inc Myocardial infarction Anti-LFA1 CD18Murine Fab′2 Pasteur-Merieux/ Allograft rejection Immunotech AntovaCD40L Humanized IgG Biogen Allograft rejection Antova CD40L HumanizedIgG Biogen SLE BTI-322 CD2 Rat IgG Medimmune Inc GvHD, Psoriasis CDP571TNF-alpha Humanized IgG4 Celltech Crohn's CDP571 TNF-alpha HumanizedIgG4 Celltech Rheumatoid Arthritis CDP850 E-selectin Humanized CelltechPsoriasis Corsevin M Fact VII Chimeric Centocor Anticoagulant D2E7TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18 HumanizedICOS Pharm Inc Multiple Sclerosis Hu23F2G CD11/18 Humanized IgG ICOSPharm Inc Stroke IC14 CD14 ICOS Pharm Inc Toxic shock ICM3 ICAM-3Humanized ICOS Pharm Inc Psoriasis IDEC-114 CD80 Primatised IDECPsoriasis Pharm/Mitsubishi IDEC-131 CD40L Humanized IDEC SLE Pharm/EisaiIDEC-131 CD40L Humanized IDEC Multiple Sclerosis Pharm/Eisai IDEC-151CD4 Primatised IgG1 IDEC Rheumatoid Arthritis Pharm/GlaxoSmith KlineIDEC-152 CD23 Primatised IDEC Pharm Asthma/Allergy Infliximab TNF-alphaChimeric IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Centocor Crohn's LDP-01 beta2-integrin Humanized IgGMillennium Inc Stroke (LeukoSite Inc.) LDP-01 beta2-integrin HumanizedIgG Millennium Inc Allograft rejection (LeukoSite Inc.) LDP-02alpha4beta7 Humanized Millennium Inc Ulcerative Colitis (LeukoSite Inc.)MAK-195F TNF alpha Murine Fab′2 Knoll Pharm, Toxic shock BASF MDX-33CD64 (FcR) Human Medarex/Centeon Autoimmune haematogical disordersMDX-CD4 CD4 Human IgG Medarex/Eisai/ Rheumatoid Arthritis GenmabMEDI-507 CD2 Humanized Medimmune Inc Psoriasis MEDI-507 CD2 HumanizedMedimmune Inc GvHD OKT4A CD4 Humanized IgG Ortho Biotech Allograftrejection OrthoClone CD4 Humanized IgG Ortho Biotech Autoimmune diseaseOKT4A Orthoclone/ CD3 Murine mIgG2a Ortho Biotech Allograft rejectionanti-CD3 OKT3 RepPro/ gpIIbIIIa Chimeric Fab Centocor/LillyComplications of Abciximab coronary angioplasty rhuMab- IgE HumanizedIgG1 Genentech/Novartis/ Asthma/Allergy E25 Tanox Biosystems SB-240563IL5 Humanized GlaxoSmithKline Asthma/Allergy SB-240683 IL-4 HumanizedGlaxoSmithKline Asthma/Allergy SCH55700 IL-5 Humanized Celltech/ScheringAsthma/Allergy Simulect CD25 Chimeric IgG1 Novartis Pharm Allograftrejection SMART CD3 Humanized Protein Design Autoimmune disease a-CD3Lab SMART CD3 Humanized Protein Design Allograft rejection a-CD3 LabSMART CD3 Humanized IgG Protein Design Psoriasis a-CD3 Lab Zenapax CD25Humanized IgG1 Protein Design Allograft rejection Lab/Hoffman- La Roche

TABLE 12 ANTIBODIES FOR AUTOIMMUNE DISORDERS THAT CAN BE ENGINEERED INACCORDANCE WITH THE INVENTION Antibody Indication Target Antigen ABX-RB2antibody to CBL antigen on T cells, B cells and NK cells fully humanantibody from the Xenomouse 5c8 (Anti CD-40 ligand Phase II trials werehalted in October 1999 CD-40 antibody) examine “adverse events” IDEC 131systemic lupus erythyematous (SLE) anti CD40 humanized IDEC 151rheumatoid arthritis primatized; anti-CD4 IDEC 152 Asthma primatized;anti-CD23 IDEC 114 Psoriasis primatized anti-CD80 MEDI-507 rheumatoidarthritis; multiple sclerosis anti-CD2 Crohn's disease Psoriasis LDP-02(anti-b7 mAb) inflammatory bowel disease a4b7 integrin receptor onChron's disease white blood cells (leukocytes) ulcerative colitis SMARTAnti-Gamma autoimmune disorders Anti-Gamma Interferon Interferonantibody Verteportin rheumatoid arthritis MDX-33 blood disorders causedby autoimmune monoclonal antibody against reactions FcRI receptorsIdiopathic Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemiaMDX-CD4 treat rheumatoid arthritis and other monoclonal antibody againstautoimmunity CD4 receptor molecule VX-497 autoimmune disorders inhibitorof inosine multiple sclerosis monophosphate rheumatoid arthritisdehydrogenase inflammatory bowel disease (enzyme needed to make newlupus RNA and DNA psoriasis used in production of nucleotides needed forlymphocyte proliferation) VX-740 rheumatoid arthritis inhibitor of ICEinterleukin-1 beta (converting enzyme controls pathways leading toaggressive immune response) VX-745 specific to inflammation inhibitor ofP38MAP kinase involved in chemical signalling of immune mitogenactivated protein response kinase onset and progression of inflammationEnbrel (etanercept) targets TNF (tumor necrosis factor) IL-8 fully humanmonoclonal antibody against IL-8 (interleukin 8) Apogen MP4 recombinantantigen selectively destroys disease associated T-cells inducesapoptosis T-cells eliminated by programmed cell death no longer attackbody's own cells specific apogens target specific T-cells

6.4.2.1 Immunomodulatory Agents and Anti-Inflammatory Agents

The present invention provides methods of treatment for autoimmunediseases and inflammatory diseases comprising administration of themolecules with variant Fc regions having an enhanced affinity forFcγRIIB and a decreased affinity for FcγRIIIA and/or FcγRIIA inconjunction with other treatment agents. Examples of immunomodulatoryagents include, but are not limited to, methotrexate, ENBREL, REMICADE™,leflunomide, cyclophosphamide, cyclosporine A, and macrolide antibiotics(e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids,steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitrilamides (e.g., leflunomide), Tcell receptor modulators, and cytokine receptor modulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINET™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumetone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

6.4.3 Infectious Disease

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylactically effective amount of one or moremolecules of the invention. Infectious diseases that can be treated orprevented by the molecules of the invention are caused by infectiousagents including but not limited to viruses, bacteria, fungi, protozae,and viruses.

Viral diseases that can be treated or prevented using the molecules ofthe invention in conjunction with the methods of the present inventioninclude, but are not limited to, those caused by hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral meningitis,encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by bacteria include, but are not limited to,mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borreliaburgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus,streptococcus, staphylococcus, mycobacterium, tetanus, pertussis,cholera, plague, diptheria, chlamydia, S. aureus and legionella.

Protozoal diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by protozoa include, but are not limited to,leishmania, kokzidioa, trypanosoma or malaria.

Parasitic diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by parasites include, but are not limited to,chlamydia and rickettsia.

According to one aspect of the invention, molecules of the inventioncomprising variant Fc regions have an enhanced antibody effectorfunction towards an infectious agent, e.g., a pathogenic protein,relative to a comparable molecule comprising a wild-type Fc region.Examples of infectious agents include but are not limited to bacteria(e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus,Enterococcus faecials, Candida albicans, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa), a pathogen (e.g.,B-lymphotropic papovavirus (LPV); Bordatella pertussis; Boma Diseasevirus (BDV); Bovine coronavirus; Choriomeningitis virus; Dengue virus; avirus, E. coli; Ebola; Echovirus 1; Echovirus-11 (EV); Endotoxin (LPS);Enteric bacteria; Enteric Orphan virus; Enteroviruses; Feline leukemiavirus; Foot and mouth disease virus; Gibbon ape leukemia virus (GALV);Gram-negative bacteria; Heliobacter pylori; Hepatitis B virus (HBV);Herpes Simplex Virus; HIV-1; Human cytomegalovirus; Human coronavirus;Influenza A, B & C; Legionella; Leishmania mexicana; Listeriamonocytogenes; Measles virus; Meningococcus; Morbilliviruses; Mousehepatitis virus; Murine leukemia virus; Murine gamma herpes virus;Murine retrovirus; Murine coronavirus mouse hepatitis virus;Mycobacterium avium-M; Neisseria gonorrhoeae; Newcastle disease virus;Parvovirus B19; Plasmodium falciparum; Pox Virus; Pseudomonas;Rotavirus; Salmonella typhiurium; Shigella; Streptococci; T-celllymphotropic virus 1; Vaccinia virus).

In a specific embodiment, molecules of the invention enhance theefficacy of treatment of an infectious disease by enhancing phagocytosisand/or opsonization of the infectious agent causing the infectiousdisease. In another specific embodiment, molecules of the inventionenhance the efficacy of treatment of an infectious disease by enhancingADCC of infected cells causing the infectious disease.

In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or additional therapeutic agents known to those skilled inthe art for the treatment and/or prevention of an infectious disease.The invention contemplates the use of the molecules of the invention incombination with antibiotics known to those skilled in the art for thetreatment and or prevention of an infectious disease. Antibiotics thatcan be used in combination with the molecules of the invention include,but are not limited to, macrolide (e.g., tobramycin (Tobi®)), acephalosporin (e.g., cephalexin (Keflex®), cephradine (Velosef®),cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime(Suprax®) or cefadroxil (Duricef®)), a clarithromycin (e.g.,clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin(EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen VeeK®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®)or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g., apramycin,arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin,undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, andspectinomycin), amphenicol antibiotics (e.g., azidamfenicol,chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics(e.g., rifamide and rifampin), carbacephems (e.g., loracarbef),carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g.,cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran,cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam,carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphenethicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin, clinafloxacin,flumequine, and grepafloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalyl-sulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

In certain embodiments, the molecules of the invention can beadministered in combination with a therapeutically or prophylacticallyeffective amount of one or more antifungal agents. Antifungal agentsthat can be used in combination with the molecules of the inventioninclude but are not limited to amphotericin B, itraconazole,ketoconazole, fluconazole, intrathecal, flucytosine, miconazole,butoconazole, clotrimazole, nystatin, terconazole, tioconazole,ciclopirox, econazole, haloprogrin, naftifine, terbinafine,undecylenate, and griseofuldin.

In some embodiments, the molecules of the invention can be administeredin combination with a therapeutically or prophylactically effectiveamount of one or more anti-viral agent. Useful anti-viral agents thatcan be used in combination with the molecules of the invention include,but are not limited to, protease inhibitors, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors and nucleoside analogs. Examples of antiviral agents includebut are not limited to zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin, as well as foscarnet,amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir,ritonavir, the alpha-interferons; adefovir, clevudine, entecavir,pleconaril.

6.5 Vaccine Therapy

The invention further encompasses using a composition of the inventionto induce an immune response against an antigenic or immunogenic agent,including but not limited to cancer antigens and infectious diseaseantigens (examples of which are disclosed infra). The vaccinecompositions of the invention comprise one or more antigenic orimmunogenic agents to which an immune response is desired, wherein theone or more antigenic or immunogenic agents is coated with a variantantibody of the invention that has an enhanced affinity to FcγRIIIA.Although not intending to be bound by a particular mechanism of action,coating an antigenic or immunogenic agent with a variant antibody of theinvention that has an enhanced affinity to FcγRIIIA, enhances the immuneresponse to the desired antigenic or immunogenic agent by inducinghumoral and cell-mediated responses. The vaccine compositions of theinvention are particularly effective in eliciting an immune response,preferably a protective immune response against the antigenic orimmunogenic agent.

In some embodiments, the antigenic or immunogenic agent in the vaccinecompositions of the invention comprises a virus against which an immuneresponse is desired. The viruses may be recombinant or chimeric, and arepreferably attenuated. Production of recombinant, chimeric, andattenuated viruses may be performed using standard methods known to oneskilled in the art. The invention encompasses a live recombinant viralvaccine or an inactivated recombinant viral vaccine to be formulated inaccordance with the invention. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In a specific embodiment, the recombinant virus is non-pathogenic to thesubject to which it is administered. In this regard, the use ofgenetically engineered viruses for vaccine purposes may require thepresence of attenuation characteristics in these strains. Theintroduction of appropriate mutations (e.g., deletions) into thetemplates used for transfection may provide the novel viruses withattenuation characteristics. For example, specific missense mutationswhich are associated with temperature sensitivity or cold adaption canbe made into deletion mutations. These mutations should be more stablethan the point mutations associated with cold or temperature sensitivemutants and reversion frequencies should be extremely low. RecombinantDNA technologies for engineering recombinant viruses are known in theart and encompassed in the invention. For example, techniques formodifying negative strand RNA viruses are known in the art, see, e.g.,U.S. Pat. No. 5,166,057, which is incorporated herein by reference inits entirety.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed for use in the intradermal vaccine formulations of theinvention. Such viruses would go through only one or a few rounds ofreplication within the host. When used as a vaccine, the recombinantvirus would go through limited replication cycle(s) and induce asufficient level of immune response but it would not go further in thehuman host and cause disease. Alternatively, inactivated (killed) virusmay be formulated in accordance with the invention. Inactivated vaccineformulations may be prepared using conventional techniques to “kill” thechimeric viruses. Inactivated vaccines are “dead” in the sense thattheir infectivity has been destroyed. Ideally, the infectivity of thevirus is destroyed without affecting its immunogenicity. In order toprepare inactivated vaccines, the chimeric virus may be grown in cellculture or in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled.

In certain embodiments, completely foreign epitopes, including antigensderived from other viral or non-viral pathogens can be engineered intothe virus for use in the intradermal vaccine formulations of theinvention. For example, antigens of non-related viruses such as HIV(gp160, gp120, gp41) parasite antigens (e.g., malaria), bacterial orfungal antigens or tumor antigens can be engineered into the attenuatedstrain.

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in the intradermal vaccineformulations. Preferably, heterologous gene sequences are moieties andpeptides that act as biological response modifiers. Preferably, epitopesthat induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention include, but are not limited to, influenza andparainfluenza hemagglutinin neuraminidase and fusion glycoproteins suchas the HN and F genes of human PIV3. In yet another embodiment,heterologous gene sequences that can be engineered into the chimericviruses include those that encode proteins with immuno-modulatingactivities. Examples of immuno-modulating proteins include, but are notlimited to, cytokines, interferon type 1, gamma interferon, colonystimulating factors, interleukin-1, -2, -4, -5, -6, -12, and antagonistsof these agents.

In yet other embodiments, the invention encompasses pathogenic cells orviruses, preferably attenuated viruses, which express the variantantibody on their surface.

In alternative embodiments, the vaccine compositions of the inventioncomprise a fusion polypeptide wherein an antigenic or immunogenic agentis operatively linked to a variant antibody of the invention that has anenhanced affinity for FcγRIIIA. Engineering fusion polypeptides for usein the vaccine compositions of the invention is performed using routinerecombinant DNA technology methods and is within the level of ordinaryskill.

The invention further encompasses methods to induce tolerance in asubject by administering a composition of the invention. Preferably acomposition suitable for inducing tolerance in a subject, comprises anantigenic or immunogenic agent coated with a variant antibody of theinvention, wherein the variant antibody has a higher affinity toFcγRIIB. Although not intending to be bound by a particular mechanism ofaction, such compositions are effective in inducing tolerance byactivating the FcγRIIB mediatated inhibitory pathway.

6.6 Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising molecules of the invention (i.e., antibodies, polypeptides)comprising variant Fc regions. The invention also provides methods oftreatment, prophylaxis, and amelioration of one or more symptomsassociated with a disease, disorder or infection by administering to asubject an effective amount of a fusion protein or a conjugated moleculeof the invention, or a pharmaceutical composition comprising a fusionprotein or a conjugated molecule of the invention. In a preferredaspect, an antibody, a fusion protein, or a conjugated molecule, issubstantially purified (i.e., substantially free from substances thatlimit its effect or produce undesired side-effects). In a specificembodiment, the subject is an animal, preferably a mammal such asnon-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey such as, a cynomolgous monkey and a human). In apreferred embodiment, the subject is a human. In yet another preferredembodiment, the antibody of the invention is from the same species asthe subject.

Various delivery systems are known and can be used to administer acomposition comprising molecules of the invention (i.e., antibodies,polypeptides), comprising variant Fc regions, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or fusion protein, receptor-mediated endocytosis(See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), constructionof a nucleic acid as part of a retroviral or other vector, etc. Methodsof administering a molecule of the invention include, but are notlimited to, parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal and oral routes). In a specific embodiment, themolecules of the invention are administered intramuscularly,intravenously, or subcutaneously. The compositions may be administeredby any convenient route, for example, by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078;and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO98/31346; and WO 99/66903, each of which is incorporated herein byreference in its entirety.

The invention also provides that the molecules of the invention (i.e.,antibodies, polypeptides) comprising variant Fc regions, are packaged ina hermetically sealed container such as an ampoule or sachetteindicating the quantity of antibody. In one embodiment, the molecules ofthe invention are supplied as a dry sterilized lyophilized powder orwater free concentrate in a hermetically sealed container and can bereconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, the moleculesof the invention are supplied as a dry sterile lyophilized powder in ahermetically sealed container at a unit dosage of at least 5 mg, morepreferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilizedmolecules of the invention should be stored at between 2 and 8° C. intheir original container and the molecules should be administered within12 hours, preferably within 6 hours, within 5 hours, within 3 hours, orwithin 1 hour after being reconstituted. In an alternative embodiment,molecules of the invention are supplied in liquid form in a hermeticallysealed container indicating the quantity and concentration of themolecule, fusion protein, or conjugated molecule. Preferably, the liquidform of the molecules of the invention are supplied in a hermeticallysealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml,at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, atleast 150 mg/ml, at least 200 mg/ml of the molecules.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of antibodies of the invention or fragments thereof maybe reduced by enhancing uptake and tissue penetration of the antibodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the molecules of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the molecules of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidmolecules are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a molecule of the invention, care must be taken touse materials to which the molecule does not absorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more molecules of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al., 1996, “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.,1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Intl Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al., 1997, “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980,Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). Inanother embodiment, polymeric materials can be used to achievecontrolled release of antibodies (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; See alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253). Examples of polymers used insustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target (e.g., the lungs), thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).In another embodiment, polymeric compositions useful as controlledrelease implants are used according to Dunn et al. (See U.S. Pat. No.5,945,155). This particular method is based upon the therapeutic effectof the in situ controlled release of the bioactive material from thepolymer system. The implantation can generally occur anywhere within thebody of the patient in need of therapeutic treatment. In anotherembodiment, a non-polymeric sustained delivery system is used, whereby anon-polymeric implant in the body of the subject is used as a drugdelivery system. Upon implantation in the body, the organic solvent ofthe implant will dissipate, disperse, or leach from the composition intosurrounding tissue fluid, and the non-polymeric material will graduallycoagulate or precipitate to form a solid, microporous matrix (See U.S.Pat. No. 5,888,533).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage and frequency ofadministration of antibodies of the invention may be reduced also byenhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of molecules of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with molecules of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the molecules used for treatment may increase or decrease overthe course of a particular treatment.

6.6.1 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of one or more molecules of theinvention and a pharmaceutically acceptable carrier.

In one particular embodiment, the pharmaceutical composition comprises atherapeutically effective amount of one or more molecules of theinvention comprising a variant Fc region, wherein said variant Fc regionbinds FcγRIIIA and/or FcγRIIA with a greater affinity than a comparablemolecule comprising a wild-type Fc region binds FcγRIIIA and/or FcγRIIAand/or said variant Fc region mediates an effector function at least2-fold more effectively than a comparable molecule comprising awild-type Fc region, and a pharmaceutically acceptable carrier. Inanother embodiment, the pharmaceutical composition comprises atherapeutically effective amount of one or more molecules of theinvention comprising a variant Fc region, wherein said variant Fc regionbinds FcγRIIIA with a greater affinity than a comparable moleculecomprising a wild-type Fc region binds FcγRIIIA, and said variant Fcregion binds FcγRIIB with a lower affinity than a comparable moleculecomprising a wild-type Fc region binds FcγRIIB, and/or said variant Fcregion mediates an effector function at least 2-fold more effectivelythan a comparable molecule comprising a wild-type Fc region, and apharmaceutically acceptable carrier. In another embodiment, saidpharmaceutical compositions further comprise one or more anti-canceragents.

The invention also encompasses pharmaceutical compositions comprising atherapeutic antibody (e.g., tumor specific monoclonal antibody) that isspecific for a particular cancer antigen, comprising one or more aminoacid modifications in the Fc region as determined in accordance with theinstant invention, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

6.6.2 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with the molecules of the invention (i.e.,antibodies, polypeptides comprising variant Fc regions). Additionally,one or more other prophylactic or therapeutic agents useful for thetreatment of a disease can also be included in the pharmaceutical packor kit. The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more molecules of theinvention. In another embodiment, a kit further comprises one or moreother prophylactic or therapeutic agents useful for the treatment ofcancer, in one or more containers. In another embodiment, a kit furthercomprises one or more cytotoxic antibodies that bind one or more cancerantigens associated with cancer. In certain embodiments, the otherprophylactic or therapeutic agent is a chemotherapeutic. In otherembodiments, the prophylactic or therapeutic agent is a biological orhormonal therapeutic.

6.7 Characterization and Demonstration Of Therapeutic Utility

Several aspects of the pharmaceutical compositions, prophylactic, ortherapeutic agents of the invention are preferably tested in vitro, in acell culture system, and in an animal model organism, such as a rodentanimal model system, for the desired therapeutic activity prior to usein humans. For example, assays which can be used to determine whetheradministration of a specific pharmaceutical composition is desired,include cell culture assays in which a patient tissue sample is grown inculture, and exposed to or otherwise contacted with a pharmaceuticalcomposition of the invention, and the effect of such composition uponthe tissue sample is observed. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. In various specific embodiments, in vitroassays can be carried out with representative cells of cell typesinvolved in an autoimmune or inflammatory disorder (e.g., T cells), todetermine if a pharmaceutical composition of the invention has a desiredeffect upon such cell types.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary. Said aspectsinclude the temporal regime of administering the prophylactic and/ortherapeutic agents, and whether such agents are administered separatelyor as an admixture.

Preferred animal models for use in the methods of the invention are, forexample, transgenic mice expressing human FcγRs on mouse effector cells,e.g., any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety) can be used in thepresent invention. Transgenic mice for use in the methods of theinvention include, but are not limited to, mice carrying human FcγRIIIA;mice carrying human FcγRIIA; mice carrying human FcγRIIB and humanFcγRIIIA; mice carrying human FcγRIIB and human FcγRIIA.

Preferably, mutations showing the highest levels of activity in thefunctional assays described above will be tested for use in animal modelstudies prior to use in humans. Antibodies harboring the Fc mutantsidentified using the methods of the invention and tested in ADCC assays,including ch4D5 and ch520C9, two anti-Erb-B2 antibodies, and chCC49, ananti-TAG72 antibody, are preferred for use in animal models since theyhave been used previously in xenograft mouse model (Hudsiak et al.,1989, Mol. Cell Biol. 9: 1165-72; Lewis et al., 1993, Cancer Immunol.Immunother. 37: 255-63; Bergman et al., 2001 Clin. Cancer Res. 7:2050-6; Johnson et al., 1995, Anticancer Res. 1387-93). Sufficientquantities of antibodies may be prepared for use in animal models usingmethods described supra, for example using mammalian expression systemsand IgG purification methods disclosed and exemplified herein. A typicalexperiment requires at least about 5.4 mg of mutant antibody. Thiscalculation is based on average quantities of wild type antibodyrequired to protect 8-10 30 g mice following a loading dose of 4 μg/gand a weekly maintenance dose, 2 μg/g, for ten weeks. inventionencompassed tumor cell lines as a source for xenograft tumors, such asSK-BR-3, BT474 and HT29 cells which are derived from patients withbreast adenocarcinoma. These cells have both Erb-B2 and the prolactinreceptors on their surface. The SK-BR-3 cells have been usedsuccessfully in both ADCC and xenograft tumor models. In other assaysOVCAR3 cells derived from a human ovarian adenocarcinoma may be used.These cells express the antigen TAG72 on the cell surface and can beused in conjunction with the chCC49 antibody. The use of differentantibodies and multiple tumor models will circumvent loss of anyspecific mutations due to an antibody specific Fc mutantincompatibility.

Mouse xenograft models may be used for examining efficacy of mouseantibodies generated against a tumor specific target based on theaffinity and specificity of the CDR regions of the antibody molecule andthe ability of the Fc region of the antibody to elicit an immuneresponse (Wu et al., 2001, Trends Cell Biol. 11: S2-9). Transgenic miceexpressing human FcγRs on mouse effector cells are unique and aretailor-made animal models to test the efficacy of human Fc-FcγRinteractions. Pairs of FcγRIIIA, FcγRIIIB and FcγRIIA transgenic mouselines generated in the lab of Dr. Jeffrey Ravetch (Through a licensingagreement with Rockefeller U. and Sloan Kettering Cancer center) can beused such as those listed in the Table 13 below.

TABLE 13 Mice Strains Strain Background Human FcR Nude/CD16A KO noneNude/CD16A KO FcγRIIIA Nude/CD16A KO FcγR IIA Nude/CD16A KO FcγR IIA andIIIA Nude/CD32B KO none Nude/CD32B KO FcγR IIB

Preferably Fc mutants showing both enhanced binding to FcγRIIIA andreduced binding to FcγRIIB, increased activity in ADCC and phagocytosisassays are tested in animal model experiments. The animal modelexperiments examine the increase in efficacy of Fc mutant bearingantibodies in FcγRIIIA transgenic, nude mCD16A knockout mice compared toa control which has been administered native antibody. Preferably,groups of 8-10 mice are examined using a standard protocol. An exemplaryanimal model experiment may comprise the following steps: in a breastcancer model, ˜2×10⁶ SK-BR-3 cells are injected subcutaneously on day 1with 0.1 mL PBS mixed with Matrigel (Becton Dickinson). Initially a wildtype chimeric antibody and isotype control are administered to establisha curve for the predetermined therapeutic dose, intravenous injection of4D5 on day 1 with an initial dose of 4 μg/g followed by weeklyinjections of 2 μg/g. Tumor volume is monitored for 6-8 weeks to measureprogress of the disease. Tumor volume should increase linearly with timein animals injected with the isotype control. In contrast very littletumor growth should occur in the group injected with 4D5. Results fromthe standard dose study are used to set an upper limit for experimentstesting the Fc mutants. These studies are done using subtherapeuticdoses of the Fc mutant containing antibodies. A one tenth dose was usedon xenograft models in experiments done in FcγRIIB knockout mice, see,Clynes et al., 2000, Nat. Med. 6: 443-6, with a resultant block in tumorcell growth. Since the mutants of the invention preferrably show anincrease in FcγRIIIA activation and reduction in FcγRIIB binding themutants are examined at one tenth therapeutic dose. Examination of tumorsize at different intervals indicates the efficacy of the antibodies atthe lower dose. Statistical analysis of the data using t test provides away of determining if the data is significant. Fc mutants that showincreased efficacy are tested at incrementally lower doses to determinethe smallest possible dose as a measure of their efficacy.

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.). Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneousanimal models of inflammatory arthritis and autoimmune rheumaticdiseases can also be used to assess the anti-inflammatory activity ofthe combination therapies of invention. The following are some assaysprovided as examples, and not by limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra P. et al., “Carrageenan-Induced Arthritis in theRat,” Inflammation, 24(2): 141-155, (2000). Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al., “Carrageenan-Induced Edema in Hind Paw of the Ratas an Assay for Anti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547, (1962). This assay has been used as a primary in vivo screenfor the anti-inflammatory activity of most NSAIDs, and is consideredpredictive of human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

Additionally, animal models for inflammatory bowel disease can also beused to assess the efficacy of the combination therapies of invention(Kim et al., 1992, Scand. J. Gastroentrol. 27:529-537; Strober, 1985,Dig. Dis. Sci. 30(12 Suppl):3S-10S). Ulcerative cholitis and Crohn'sdisease are human inflammatory bowel diseases that can be induced inanimals. Sulfated polysaccharides including, but not limited toamylopectin, carrageen, amylopectin sulfate, and dextran sulfate orchemical irritants including but not limited to trinitrobenzenesulphonicacid (TNBS) and acetic acid can be administered to animals orally toinduce inflammatory bowel diseases.

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus erythematosus, and glomerulonephritis have been developed(Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999,Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

Preferred animal models for determining the therapeutic efficacy of themolecules of the invention are mouse xenograft models. Tumor cell linesthat can be used as a source for xenograft tumors include but are notlimited to, SKBR3 and MCF7 cells, which can be derived from patientswith breast adenocarcinoma. These cells have both erbB2 and prolactinreceptors. SKBR3 cells have been used routinely in the art as ADCC andxenograft tumor models. Alternatively, OVCAR3 cells derived from a humanovarian adenocarcinoma can be used as a source for xenograft tumors.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

7. EXAMPLES

7.1 Analysis of Kinetic Parameters of Fc Mutants

Fc mutants exhibiting altered affinity to FcγRIIIA and FcγRIIB weredetermined from yeast display technology and FcγR-Fc interaction assaysas disclosed in U.S Patent Application Publications 2005/0037000 and2005/0064514, and International Patent Application Publication WO04/063351, each of which is hereby incorporated by reference in itsentirety. Effects of the mutant in in vitro assays was assessed bydetermining kinetic parameters of the binding of ch4-4-20 antibodiesharboring the Fc mutants using a BIAcore assay (BIAcore instrument 1000,BIAcore Inc., Piscataway, N.J.). The FcγRIIIA used in this assay was asoluble monomeric protein, the extracellular region of FcγRIIIA joinedto the linker-AVITAG sequence, while the FcγRIIB used in this assay wasa soluble dimeric protein, both were prepared as described in U.S PatentApplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351. Briefly, theFcγRIIB used was the extracellular domain of FcγRIIB fused to thehinge-CH2-CH3 domain of human IgG2.

BSA-FITC (36 μg/mL in 10 mM Acetate Buffer at pH 5.0) was immobilized onone of the four flow cells (flow cell 2) of a sensor chip surfacethrough amine coupling chemistry (by modification of carboxymethylgroups with mixture of NHS/EDC) such that about 5000 response units (RU)of BSA-FITC was immobilized on the surface. Following this, theunreacted active esters were “capped off” with an injection of 1MEt-NH2. Once a suitable surface was prepared, ch 4-4-20 antibodiescarrying the Fc mutations were passed over the surface by one minuteinjections of a 20 μg/mL solution at a 5 μL/mL flow rate. The level ofch-4-4-20 antibodies bound to the surface ranged between 400 and 700 RU.Next, dilution series of the receptor (FcγRIIIA and FcγRIIB-Fc fusionprotein) in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005% SurfactantP20, 3 mM EDTA, pH 7.4) were injected onto the surface at 100 μL/min.Antibody regeneration between different receptor dilutions was carriedout by single 5 second injections of 100 mM NaHCO₃ pH 9.4; 3M NaCl.

The same dilutions of the receptor were also injected over a BSA-FITCsurface without any ch-4-4-20 antibody at the beginning and at the endof the assay as reference injections.

Once an entire data set was collected, the resulting binding curves wereglobally fitted using computer algorithms supplied by the manufacturer,BIAcore, Inc. (Piscataway, N.J.). These algorithms calculate both theK_(on) and K_(off), from which the apparent equilibrium bindingconstant, K_(D) is deduced as the ratio of the two rate constants (i.e.,K_(off)/K_(on)). More detailed treatments of how the individual rateconstants are derived can be found in the BIAevaluation SoftwareHandbook (BIAcore, Inc., Piscataway, N.J.).

Binding curves for two different concentrations (200 nM and 800 nM forFcγRIIIA and 200 nM and 400 nM for FcγRIIB fusion protein) were alignedand responses adjusted to the same level of captured antibodies, and thereference curves were subtracted from the experimental curves.Association and dissociation phases were fitted separately. Dissociationrate constant was obtained for interval 32-34 sec of the dissociationphase; association phase fit was obtained by a 1:1 Langmuir model andbase fit was selected on the basis R_(max) and chi² criteria.

Results

FIG. 4 shows the capture of ch 4-4-20 antibodies with mutant Fc regionson the BSA-FTIC-immobilized sensor chip. 64 of antibodies at aconcentration of about 20 μg/mL were injected at 5 μL/min over theBSA-FITC surface. FIG. 5 is a sensogram of real time binding of FcγRIIIAto ch-4-4-20 antibodies carrying variant Fc regions. Binding of FcγRIIIAwas analyzed at 200 nM concentration and resonance signal responses werenormalized at the level of the response obtained for the wild typech-4-4-20 antibody. Kinetic parameters for the binding of FcγRIIIA toch-4-4-20 antibodies were obtained by fitting the data obtained at twodifferent FcγRIIIA concentrations, 200 and 800 nM (FIG. 6). The solidline represents the association fit which was obtained based on theK_(off) values calculated for the dissociation curves in interval 32-34seconds. K_(D) and K_(off) represent the average calculated from the twodifferent FcγRIIIA concentrations used. FIG. 7 is a sensogram of realtime binding of FcγRIIB-Fc fusion protein to ch-4-4-20 antibodiescarrying variant Fc regions. Binding of FcγRIIB-Fc fusion protein wasanalyzed at 200 nM concentration and resonance signal responses werenormalized at the level of the response obtained for the wild typech-4-4-20 antibody. Kinetic parameters for the binding of FcγRIIB-Fcfusion protein to ch-4-4-20 antibodies were obtained by fitting the dataobtained at two different FcγRIIB-Fc fusion protein concentrations, 200and 800 nM (FIG. 8). The solid line represents the association fit whichwas obtained based on the K_(off) calculated for the dissociation curvesin interval 32-34 seconds. K_(D) and K_(off) represent the average fromthe two different FcγRIIB-Fc fusion protein concentrations used.

The kinetic parameters (K_(on) and K_(off)) that were determined fromthe BIAcore analysis correlated with the binding characteristic of themutants as determined by an ELISA assay and the functional activity ofthe mutants as determined in an ADCC assay. Specifically, as seen inTable 14, mutants that had an enhanced ADCC activity relative to thewild-type protein, and had an enhanced binding to FcγRIIIA as determinedby an ELISA assay had an improved K_(off) for FcγRIIIA (i.e., a lowerK_(off)). Therefore, a lower K_(off) value for FcγRIIIA for a mutant Fcprotein relative to a wild type protein may be likely to have anenhanced ADCC function. On the other hand, as seen in Table 15, mutantsthat had an enhanced ADCC activity relative to the wild-type protein,and had a reduced binding for FcγRIIB-Fc fusion protein as determined byan ELISA assay had a higher K_(off) for FcγRIIB-Fc fusion protein.

Thus, the K_(off) values for FcγRIIIA and FcγRIIB can be used aspredictive measures of how a mutant will behave in a functional assaysuch as an ADCC assay. In fact, ratios of K_(off) values for FcγRIIIAand FcγRIIB-Fc fusion protein of the mutants to the wild type proteinwere plotted against ADCC data (FIG. 9). Specifically, in the case ofK_(off) values for FcγRIIIA, the ratio of K_(off) (wt)/K_(off) (mutant)was plotted against the ADCC data; and in the case of K_(off) values forFcγRIIB, the ratio of K_(off) (mut)/K_(off) (wt) was plotted against theADCC data. Numbers higher than one (1) show a decreased dissociationrate for FcγRIIIA and an increased dissociation rate for FcγRIIB-Fcrelative to wild type. Mutants that fall within the indicated box have alower off rate for FcγRIIIA binding and a higher off-rate for FcγRIIB-Fcbinding, and possess an enhanced ADCC function.

TABLE 14 Kinetic parameters of FcRIIIA binding to ch4-4-20Ab obtained by“separate fit” of 200 nM and 800 nM binding curves BIAcore K_(on)Ch4-4-20Ab Kd, nM 1/Ms K_(off), 1/s ELISA, OD ADCC, % Wt(0225) 319  6.0× 10⁵ 0.170 0.5 17.5 Mut11(0225) 90 8.22 × 10⁵ 0.075 0.37 32 Mut5(0225)214  8.2 × 10⁵ 0.172 0.75 26 Mut6(0225) 264 6.67 × 10⁵ 0.175 0.6 23Mut8(0225) 234  8.3 × 10⁵ 0.196 0.5 22 Mut10(0225) 128 9.04 × 10⁵ 0.1151.0 41 Mut12(0225) 111 1.04 × 10⁶ 0.115 1.0 37 Mut15(0225) 67.9 1.97 ×10⁶ 0.133 1.0 15 Mut16(0225) 84.8 1.60 × 10⁶ 0.133 1.0 15 Mut18(0225) 921.23 × 10⁶ 0.112 1.0 28 Mut25(0225) 48.6 2.05 × 10⁶ 0.1 1.0 41Mut14(0225) 75.4 1.37 × 10⁶ 0.1 1.1 28 Mut17(0225) 70.5 1.42 × 10⁶ 0.11.25 30 Mut19(0225) 100 1.20 × 10⁶ 0.120 0.75 11 Mut20(0225) 71.5 1.75 ×10⁶ 0.126 0.5 10 Mut23(0225) 70.2 1.43 × 10⁶ 0.105 1.25 25Highlighted mutants do not fit to the group by ELISA or ADCC data.

TABLE 15 Kinetic parameters of FcRIIB-Fc binding to wild type and mutantch4-4-20Ab obtained by “separate fit” of 200 nM and 800 nM bindingcurves. BIAcore K_(on) Ch4-4-20Ab Kd, nM 1/Ms K_(off), 1/s ELISA, ODADCC, % Wt(0225) 61.4 0.085 0.4 17.5 Mut11(0225) 82.3 0.1 0.08 32Mut5(0225) 50 0.057 0.6 26 Mut6(0225) 66.5 0.060 0.35 23 Mut8(0225) 44.20.068 0.25 22 Mut10(0225) 41.3 0.05 1.2 41 Mut12(0225) 40.1 0.051 0.4 37Mut15(0225) 37.8 0.040 1.55 15 Mut16(0225) 40 0.043 1.55 15 Mut18(0225)51.7 0.043 1.25 28 Mut25(0225) 0.112 0.08 41 Mut14(0225) 95.6 0.089 0.1328 Mut17(0225) 55.3 0.056 0.38 30 Mut19(0225) 45.3 0.046 1.0 11Mut20(0225) 24.1 0.028 0.8 10 Mut23(0225) 108 0.107 0.1 25

7.2 Screening for Fc Mutants Using Multiple Rounds of Enrichment Using aSolid Phase Assay

The following mutant screens were aimed at identifying additional setsof mutants that show improved binding to FcγRIIIA and reduced binding toFcγRIIB. Secondary screening of selected Fc variants was performed byELISA followed by testing for ADCC in the 4-4-20 system. Mutants werethan selected primarily based on their ability to mediate ADCC via4-4-20 using Fluorescein coated SK-BR3 cells as targets and isolatedPBMC from human donors as the effector cell population. Fc mutants thatshowed a relative increase in ADCC, e.g., an enhancement by a factor of2 were than cloned into anti-HER2/neu or anti-CD20 chAbs and tested inan ADCC assay using the appropriate tumor cells as targets. The mutantswere also analyzed by BIAcore and their relative K_(off) weredetermined.

Screen 1: Sequential Solid Phase Depletion and Selection Using MagneticBeads Coated with FcγRIIB Followed by Selection with Magnetic BeadsCoated with FcγRIIIA.

The aim of this screen was identification of Fc mutants that either nolonger bind FcγRIIB or show reduced binding to FcγRIIB. A 10-fold excessof the naïve library (˜10⁷ cells) was incubated with magnetic beads (“MyOne”, Dynal) coated with FcγRIIB. Yeast bound to beads were separatedfrom the non-bound fraction by placing the tube containing the mixturein a magnetic field. Those yeast cells that were not bound to the beadswere removed and placed in fresh media. They were next bound to beadsthat were coated with FcγRIIIA. Yeast bound to beads were separated fromthe nonbound fraction by placing the tube containing the mixture in amagnetic field. Nonbound yeast were removed and the bound cells wereremoved by vigorous vortexing. The recovered cells were regrown inglucose containing media and reinduced in selective media containinggalactose. The selection process was repeated. The final culture wasthan used to harvest DNA. Inserts containing the Fc domain wereamplified by PCR and cloned into 4-4-20. Approximately 90 Fc mutantswere screened by 4-4-20 ELISA and ADCC assays and the resultant positivemutants are shown in Table 16.

TABLE 16 Mutants selected by sequential solid phase depletion andselection using Magnetic beads coated with FcγRIIB followed by selectionwith magnetic beads coated with FcγRIIIA. Mutant Amino Acid changesMgFc37 K248M MgFc38 K392T, P396L MgFc39 E293V, Q295E, A327T MgFc41H268N, P396LN MgFc43 Y319F, P352L, P396L MgFc42 D221E, D270E, V308A,Q311H, P396L, G402D

Screens 2&3: Mutants Selected by FACS, Equilibrium and KineticScreening:

The first library screen identified a mutation at position 396, changingthe amino acid from Proline to Leucine (P396L). This Fc variant showedincreased binding to both FcγRIIIA and FcγRIIB. A second library wasconstructed using P396L as a base line. PCR mutagenesis was used togenerate ˜10⁷ mutants each of which contained the P396L mutation andcontained additional nucleotide changes. The P396L library was screenedusing two sets of conditions.

An equilibrium screen was performed using biotinylatedFcγRIIIA-linker-avitag as a monomer, using methods already described.Approximately 10-fold excess of library (10⁸ cells) was incubated in a0.5 mL of approximately 7 nM FcγRIIIA for 1 hr. The mixture was sortedby FACS, selecting top 1.2% of binders. Selected yeast cells were grownin selective media containing glucose and reinduced in selective mediacontaining galactose. The equilibrium screen was repeated a second timeand the sort gate was set to collect the top 0.2% of binders. Theselected yeast cells were then grown under selective conditions inglucose. This culture was than used to harvest DNA. Inserts containingthe Fc domain were amplified by PCR and cloned into the nucleotidesequence encoding 4-4-20 variable domain using methods alreadydescribed. Approximately 90 Fc mutants were screened by 4-4-20 ELISA andADCC and the resultant positive mutants are shown in Table 17.

TABLE 17 Mutants selected by FACS using an Equilibrium screen withconcentrations of FcRIIIA of approximately 7 nM. Mutant Amino Acidchanges MgFc43b K288R, T307A, K344E, P396L MgFc44 K334N, P396L MgFc46P217S, P396L MgFc47 K210M, P396L MgFc48 V379M, P396L MgFc49 K261N,K210M, P396L MgFc60 P217S, P396L

A kinetic screen was also implemented to identify mutants with improvedK_(off) in binding FcγRIIIA. Conditions were established for screeningthe P396L library using a strain with the P396L Fc variant displayed onthe yeast surface. Briefly cells grown under inducing conditions wereincubated with 0.1 μM biotinylated FcγRIIIA-linker-avitag monomer for 1hr. The cells were washed to remove the labeled ligand. Labeled cellswere then incubated for different times with 0.1 μM unlabeledFcγRIIIA-linker-avitag monomer, washed and then stained with SA:PE forFACS analysis (FIG. 10). Cells were also stained with goat anti-human Fcto show that the Fc display was maintained during the experiment.

Based on the competition study it was determined that a 1 minuteincubation resulted in approximately 50% loss of cell staining. Thistime point was chosen for the kinetic screen using the P396L library.Approximately 10-fold excess of library (10⁸ cells) was incubated with0.1 μM biotinylated FcγRIIIA-linker-avitag monomer in a 0.5 mL volume.Cells were washed and then incubated for 1 minute with unlabeled ligand.Subsequently the cells were washed and labeled with SA:PE. The mixturewas sorted by FACS, selecting the top 0.3% of binders. Selected yeastcells were grown in selective media containing glucose and reinduced inselective media containing galactose. The kinetic screen was repeated asecond time and the sort gate was set to collect the top 0.2% ofbinders. The nonselected P396L library was compared to the yeast cellsselected for improved binding by FACS (FIG. 11). The histograms show thepercentage of cells that are costained with both FcγRIIIA/PE and goatanti-human Fc/FITC (upper right).

The selected yeast cells from the second sort were then grown underselective conditions in glucose. This culture was than used to harvestDNA. Inserts containing the Fc domain were amplified by PCR and clonedinto the nucleotide sequence encoding 4-4-20 variable domain usingmethods described above. Approximately 90 Fc mutants were screened by4-4-20 ELISA and ADCC and the resultant positive mutants are shown inTable 18.

TABLE 18 Mutants selected by FACS using a Kinetic screen using equimolaramounts of unlabeled CD16A for 1 minute. Mutants Amino Acid changesMgFc50 P247S, P396L MgFc51 Q419H, P396L MgFc52 V240A, P396L MgFc53L410H, P396L MgFc54 F243L, V305I, A378D, F404S, P396L MgFc55 R255l,P396L MgFc57 L242F, P396L MgFc59 K370E, P396L

Screens 4 and 5: Combining the Solid Phase FcγRIIB Depletion Step withFcγRIIIA Selection by FACs Sort, Using the FcγRIIIA 158V Allele

Analysis of Fc variants from Screen 1 showed that the mutations thatwere selected from the secondary screen had improved binding to bothFcγRIIIA and FcγRIIB. Therefore, the data suggested that sequentialdepletion and selection using magnetic beads (solid phase) under theestablished conditions did not efficiently select for differentialbinding of FcγRIIIA and FcγRIIB. Therefore, in order to screen moreeffectively for mutants that bind FcγRIIIA, while having reduced or nobinding to FcγRIIB, the solid phase FcγRIIB depletion step was combinedwith FcγRIIIA selection by FACs sort. This combination identified Fcvariants that bind FcγRIIIA with greater or equal affinity thanwild-type Fc.

A 10-fold excess of the naïve library (˜10⁷) was incubated with magneticbeads coated with FcγRIIB. Yeast bound to beads were separated from thenon-bound fraction by placing the tube containing the mixture in amagnetic field. Those yeast cells that were not bound to the beads wereremoved and placed in fresh media and subsequently reinduced in mediacontaining galactose. The FcγRIIB depletion by magnetic beads wasrepeated 5 times. The resulting yeast population was analyzed and foundto show greater than 50% cell staining with goat anti-human Fc and avery small percentage of cells were stained with FcγRIIIA. These cellswere then selected twice by a FACS sort using 0.1 μM biotinylatedFcγRIIIA linker-avitag (data not shown). The FcγRIIIA was the 158Vallotype. Yeast cells were analyzed for both FcγRIIIA and FcγRIIBbinding after each sort and compared to binding by wild-type Fc domain(FIGS. 12 A-B).

The selected yeast cells from the second sort were then grown underselective conditions in glucose. This culture was then used to harvestDNA. Inserts containing the Fc domain were amplified by PCR and clonedinto the nucleotide sequence encoding 4-4-20 variable domain.Approximately 90 Fc mutants were screened by 4-4-20 ELISA and ADCC andthe resultant positive mutants are shown in Table 19 (mutants 61-66).

TABLE 19 Mutants selected by magnetic bead depletion using beads coatedwith CD32B and final selection by FACS using FcγRIIIA 158Valine or158Phenylalanine Mutants Amino Acid Changes MgFc61 A330V MgFc62 R292GMgFc63 S298N, K360R, N361D MgFc64 E233G MgFc65 N276Y MgFc66 A330V, V427MMgFc67 V284M, S298N, K334E, R355W, R416T

Screening of Fc Mutants Using the 158F Allele of FcγRIIIA:

Two different alleles of FcγRIIIA receptor exist that have differentbinding affinities for the IgG1 Fc domain (Koene et al., 1997, Blood 90:1109-1114; Wu et al., 1997, J. Clin. Invest. 100: 1059-70). The 158Fallele binds to the Fc domain with a binding constant 5-10 fold lowerthan the 158V allele. Previously all of the Fc screens using yeastdisplay were done using the high binding 158V allele as a ligand. Inthis experiment, Fc mutants were selected from the FcγRIIB depletedyeast population using biotinylated FcγRIIIA158F-linker-avitag monomeras a ligand. The sort gate was set to select the top 0.25 percentFcγRIIIA 158F binders. The resulting enriched population was analyzed byFACS (FIG. 12B). Individual clones were then isolated and their bindingto different FcγRs were analyzed by FACS (FIG. 12B). Analysis ofindividual clones from the population resulted in the identification ofa single mutant harboring 5 mutations MgFc67 (V284M, S298N, K334E,R355W, R416S), which had an enhanced binding to FcγRIIIA and a reducedbinding to FcγRIIB.

Secondary Screen of Mutants by an ADCC Assay For Screens 1, 2, and 3:

Mutants that were selected in the above screens were then analyzed usinga standard ADCC assay to determine the relative rates of lysis mediatedby ch4-4-20 harboring the Fc mutants. ch4-4-20 antibodies carrying theFc variants were constructed using methods already described above.SK-BR3 cells were used as targets and effector cells were PBMC that wereisolated from donors using a Ficoll gradient, as described supra(Section 7.7). The ADCC activity results for the mutants are summarizedin Table 20.

As seen in Table 20, mutants isolated using the above primary andsecondary screens based on FcγRIIB depletion and FcγRIIIA selectionshowed enhanced ADCC activity relative to wild-type.

TABLE 20 Analysis of ADCC mediated by 4-4-20 anti-Fluorescein antibodyon SKBR3 cells coated with fluorescein. Relative rate of Mutant AminoAcid Change lysis MgFc37 K248M 3.83 MgFc38 K392T, P396L 3.07 MgFc39E293V, Q295E, A327T 4.29 MgFc41 H268N, P396LN 2.24 MgFc43 Y319F, P352L,P396L 1.09 MgFc42 D221E, D270E, V308A, Q311H, P396L, G402D 3.17 MgFc43bK288R, T307A, K344E, P396L 3.3 MgFc44 K334N, P396L 2.43 MgFc46 P217S,P396L 2.04 MgFc47 K210M, P396L 2.02 MgFc48 V379M, P396L 2.01 MgFc49K261N, K210M, P396L 2.06 MgFc50 P247S, P396L 2.1 MgFc51 Q419H, P396L2.24 MgFc52 V240A, P396L 2.35 MgFc53 L410H, P396L 2 MgFc54 F243L, V305I,A378D, F404S, P396L 3.59 MgFc55 R255l, P396L 2.79 MgFc57 L242F, P396L2.4 MgFc59 K370E, P396L 2.47 MgFc60 P217S, P396L 1.44

Mutants 37, 38, 39, 41, 43 were analyzed using 0.5 μg/mL ch4-4-20. Allother antibodies were tested at 1 μg/mL. All rates were normalized towild type ch4-4-20 (IgG1).

Mutants were additionally cloned into the heavy chain of antitumormonoclonal antibody 4D5 (anti-HER2/neu) and anti-CD20 monoclonalantibody 2H7 by replacing the Fc domain of these monoclonal antibodies.These chimeric monoclonal antibodies were expressed and purified andtested in an ADCC assay using standard methods by transient transfectioninto 293H cells and purification over protein G column. The chimeric 4D5antibodies were tested in an ADCC assay using SK-BR3 cells as targets(FIG. 13), whereas the chimeric 2H7 antibodies were tested in an ADCCassay using Daudi cells as targets (FIG. 14).

Secondary Screen of Mutants Via Biacore:

Mutants that were selected in the above screens were then analyzed byBIAcore to determine the kinetic parameters for binding FcγRIIIA (158V)and FcγRIIB. The method used was similar to that disclosed in Section7.1, supra.

The data displayed are K_(off) values relative to wild type off rates asdetermined from experiments using the Fc mutants in the ch4-4-20monoclonal antibody. Relative numbers greater than one indicate adecrease in K_(off) rate. Numbers less than one indicate an increase inoff rate.

Mutants that showed a decrease in off rates for FcγRIIIA were MgFc38(K392, P396L), MgFc43(Y319F, P352L, P396L), MgFc42(D221E, D270E, V308A,Q311H, P396L, G402D), MgFc43b (K288R, T307A, K344E, P396L), MgFc44(K334N, P396L), MgFc46 (P217S, P396L), MgFc49 (K261N, K210M, P396L).Mutants that showed a decrease in off rate for FcγRIIB were,MgFc38(K392, P396L), MgFc39 (E293V, Q295E, A327T), MgFc43 (K288R, T307A,K344E, P396L), MgFc44 (K334N, P396L). The Biacore data is summarized inTable 21.

TABLE 21 BIAcore data FcγRIIIA158V FcγRIIB Fc mutant AA residues(K_(off) WT/Mut) (K_(off) WT/Mut) MgFc37 K248M 0.977 1.03 MgFc38 K392T,P396L 1.64 2.3 MgFc39 E293V, Q295E, 0.86 1.3 A327T MgFc41 H268N, P396LN0.92 1.04 MgFc43 Y319F, P352L, 1.23 2.29 P396L MgFc42 D221E, D270E, 1.38V308A, Q311H, P396L, G402D MgFc43b K288R, T307A, 1.27 0.89 K344E, P396LMgFc44 K334N, P396L 1.27 1.33 MgFc46 P217S, P396L 1.17 0.95 MgFc47K210M, P396L MgFc48 V379M, P396L MgFc49 K261N, K210M, 1.29 0.85 P396LMgFc50 P247S, P396L MgFc51 Q419H, P396L MgFc52 V240A, P396L MgFc53L410H, P396L MgFc54 F243L, V305I, A378D, F404S, P396L MgFc55 R255I,P396L MgFc57 L242F, P396L MgFc59 K370E, P396L MgFc60 P217S, P396L MgFc61A330V 1 0.61 MgFc62 R292G 1 0.67 MgFc63 S298N, K360R, 1 0.67 N361DMgFc64 E233G 1 0.54 MgFc65 N276Y 1 0.64 MgFc66 A330V, G427M, 1 0.62MgFc67 V284M, S298N, K334E, R355W, R416T

7.3 PBMC Mediated ADCC Assays

Materials and Methods

Fc variants that show improved binding to FcγRIIIA were tested by PBMCbased ADCC using 60:1 effector:target ratio. Two different tumor modelsystems were used as targets, SK-BR3 (anti-HER2/neu) and Daudi(anti-CD20). Percent specific Lysis was quantitated for each mutant.Linear regression analysis was used to plot the data setting the maximalpercent lysis at 100%.

ADCC is activated on immune system effector cells via a signaltransduction pathway that is triggered by an interaction between lowaffinity FcγR and an immune complex. Effector cell populations werederived from either primary blood or activated monocyte derivedmacrophages (MDM). Target cells were loaded with europium and incubatedwith chimeric MAb and subsequently incubated with effector cellpopulations. Europium works the same way as ⁵¹Cr, but it isnon-radioactive and the released europium is detected in a fluorescentplate reader. Lymphocytes harvested from peripheral blood of donors(PBM) using a Ficoll-Paque gradient (Pharmacia) contain primarilynatural killer cells (NK). The majority of the ADCC activity will occurvia the NK containing FcγRIIIA but not FcγRIIB on their surface.

Experiments were performed using two different target cell populations,SK-BR-3 and Daudi, expressing HER2/neu and CD20, respectively. ADCCassays were set up using Ch4-4-20/FITC coated SK-BR-3, Ch4D5/SKBR3, andRituxan/Daudi (data not shown). Chimeric MAbs were modified using Fcmutations identified. Fc mutants were cloned into ch4D5. Purified Ab wasused to opsonize SK-BR-3 cells or Daudi cells. Fc mutants were clonedinto ch4D5.

Results.

Fc mutants showed improved PBMC mediated ADCC activity in SK BR3 cells(FIG. 13). The plot shows linear regression analysis of a standard ADCCassay. Antibody was titrated over 3 logs using an effector to targetratio of 75:1. % lysis=(Experimental release−SR)/(MR−SR)*100.

Fc mutants showed improved PBMC mediated ADCC activity in Daudi cells(FIG. 14).

7.4 Monocyte Derived Macrophage (MDM) Based ADCC Assays

FcγR dependent tumor cell killing is mediated by macrophage and NK cellsin mouse tumor models (Clynes et al., 1998, PNAS USA, 95: 652-6).Elutriated monocytes from donors were used as effector cells to analyzethe efficiency Fc mutants to trigger cell cytotoxicity of target cellsin ADCC assays. Expression patterns of FcγRI, FcγR3A, and FcγR2B areaffected by different growth conditions. FcγR expression from frozenmonocytes cultured in media containing different combinations ofcytokines and human serum were examined by FACS using FcR specific MAbs.(FIG. 15). Cultured cells were stained with FcγR specific antibodies andanalyzed by FACS to determine MDM FcγR profiles. Conditions that bestmimic macrophage in vivo FcγR expression, i.e., showed the greatestfraction of cells expressing CD16 and CD32B were used in a monocytederived macrophage (MDM) based ADCC assay. For the experiment in FIG.15, frozen elutriated monocytes were grown for 8 days in DMEM and 20%FBS containing either M-CSF (condition 1) or GM-CSF (condition 2). Forthe experiment in FIG. 16, frozen elutriated monocytes were cultured for2 days in DMEM and 20% FBS containing GM-CSF, IL-2 and IFNγ prior toADCC assay. Serum free conditions have also been developed which allowfor high levels of CD16 and CD32B expression (data not shown). Briefly,purified monocytes were grown for 6-8 days in Macrophage-SFM(Invitrogen) containing GM-CSF, M-CSF, IL-6, IL-10, and IL-1β. While theincidence of CD32B+/CD16+ cells in these cultures is highest using amixture of cytokines, combinations of two of more cytokines will alsoenhance FcγR expression (M-CSF/IL-6, M-CSF/IL-10; or M-CSF/IL-1β). ForADCC assays, IFNγ is added for the final 24-48 hours.

MDM based ADCC required incubation times of >16 hrs to observe targetcell killing. Target cells were loaded with Indium-111 which is retainedfor long incubations within the target cells. Indium release wasquantitated using a gamma counter. All other reagents, Abs and targetcells, were similar to the PBMC based ADCC assay. ADCC activity due toFcγRI can be efficiently blocked using the anti-FcRI blocking antibody(M21, Ancell). The assay conditions differ slightly from the PBMC basedassay. 20:1 target to effector; 18-14 hr incubation at 37 C.

Fc mutants that show improved PBMC ADCC, increased binding to FcγRIIIA,or decreased binding to FcγRIIB were tested (FIG. 16).

7.5 Effect of Fc Mutants on Complement Activity

Fc mutants were originally identified based on their increased bindingto FcγRIIIA. These mutants were subsequently validated for theirimproved affinity for all low affinity receptors and in many casesimproved activity in ADCC mediated by PBMC. In vivo antibody mediatedcytotoxicity can occur through multiple mechanisms. In addition to ADCCother possible mechanisms include complement dependent cytotoxicity(CDC) and apoptosis. The binding of C1q to the Fc region of animmunoglobulin initiates as cascade resulting in cell lysis by CDC. Theinteraction between C1q and the Fc has been studied in a series of Fcmutants. The results of these experiments indicate that C1q and the lowaffinity FcR bind to overlapping regions of the Fc, however the exactcontact residues within the Fc vary.

Mutants that showed improved ADCC in the PBMC based assay were examinedfor their effect in CDC. Antibodies were analyzed in the anti CD20Ch-mAb, 2H7. We detected improved CDC for each mutant ch-mAb tested.Interestingly even though these mutants were selected for their improvedADCC they also show enhanced CDC

Materials and Methods.

CDC assay was used to test the Fc mutants using anti-CD20 and Daudicells as targets. Guinea Pig Serum was used as the source for complement(US Biological). The CDC assay was similar to PBMC based ADCC. Targetcells were loaded with europium and opsonized with ChMAb. Howevercomplement, guinea pig serum, was added instead of effector cells. FIG.17 shows a flow chart of the assay. Anti-CD20 ChMab over 3 orders ofmagnitude was titrated. % lysis was calculated. Daudi cells, (3×10⁶)were labeled with BADTA reagent. 1×10⁴ cells were aliquoted into wellsin a 96 well plate. Antibodies were titrated into the wells using 3 folddilutions. The opsonization reaction was allowed to proceed for 30-40minutes at 37° C. in 5% CO₂. Guinea pig serum was added to a final conc.of 20%. The reaction was allowed to proceed for 3.5 hrs at 37° C. in 5%CO₂ Subsequently, 100 uls of cell media was added to the reaction andcells were spun down. For detection 20 uls of the supernatant was addedto 200 uls of the Europium solution and the plates were read in theVictor2(Wallac).

Results:

All mutants that show improved binding for either activating FcR or C1qwere placed in the CDC assay (FIG. 18). Fc mutants that showed enhancedbinding to FcγRIIIA also showed improved complement activity Each of themutants show enhanced complement activity compared to wild type. Themutants tested are double mutants. In each case one of the mutationspresent is P396L.

To determine whether the increase in CDC correlated with increasedbinding of C1q to IgG1 Fc binding between the two proteins was measuredin realtime using surface plasmon resonance. In order to examine thebinding between C1q and an IgG1 Fc the Fc variants were cloned into ananti-CD32B ch-mAb, 2B6. This allowed us to capture the wt and mutantantibodies to the glass slide via soluble CD32B protein (FIG. 19A).Three of the four mutants tested in CDC were also examined in theBiacore. All 3 showed greatly enhanced K_(off) compare to wild type Fc(FIG. 19B). BIAcore format for C1q binding to 2B6 mutants demonstrateenhanced binding of mutants with P396L mutation (FIG. 20). MutationD270E can reduce C1q binding at different extent. A summary of thekinetic analysis of FcγR and C1q binding is depicted in the Table 22below.

TABLE 22 KINETIC ANALYSIS OF FcgR and C1q binding to mutant 2B62B6Mutants 3aV158 3aF158 2bfcagl 2aR131Fcagl 2aH131Fcagl C1q WT 0.1920.434 0.056 0.070 0.053 0.124 MgFc38 0.114 0.243 0.024 0.028 0.024 0.096(K392T, P396L) MgFc51 0.142 0.310 0.030 0.036 0.028 0.074 (Q419H, P396L)MgFc55 0.146 0.330 0.030 0.034 0.028 0.080 (R255I, P396L) MgFc59 0.1490.338 0.028 0.033 0.028 0.078 (K370E, P396L) MgFc31/60 0.084 0.238 0.0940.127 0.034 0.210 MgFc51/60 0.112 0.293 0.077 0.089 0.028 0.079MgFc55/60 0.113 0.288 0.078 0.099 0.025 0.108 MgFc59/60 0.105 0.2960.078 0.095 0.024 0.107

7.6 Designing Fc Variants with Decreased Binding to FcγRIIB

Based on a selection for Fc mutants that reduce binding to FcγRIIB andincrease binding to FcγRIIA 131H a number of mutations including D270Ewere identified. Each mutation was tested individually for binding tothe low affinity Fc receptors and their allelic variants.

D270E had the binding characteristics that suggested it wouldspecifically reduce FcγRIIB binding. D270E was tested in combinationwith mutations that were previously identified based on their improvedbinding to all FcR.

Results.

As shown in Tables 23 and 24 and FIGS. 21 and 22 addition of D270Emutation enhances FcγRIIIA and FcγRIIA H131 binding and reduces bindingto FcγRIIB. FIG. 23 shows the plot of MDM ADCC data against the K_(off)as determined for CD32A H131H binding for select mutants.

TABLE 23 OFF RATE (1/s) of FcγR BINDING TO WILD TYPE AND MUTANT CHIMERIC4D5 Ab OBTAINED BY BIACORE ANALYSIS 4D5 Mutants 3aV158 3aF158 2bfcagl2aR131Fcagl 2aH131Fcagl Wt pure 0.175 0.408 0.078 0.067 0.046 MgFc550.148 0.381 0.036 0.033 0.029 MgFc55/60 0.120 0.320 0.092 0.087 0.013MgFc55/60 + 0.116 0.405 0.124 0.112 0.037 R292G MgFc55/60 + 0.106 0.3040.092 0.087 0.015 Y300L MgFc52 0.140 0.359 0.038 0.040 0.026 MgFc52/600.122 0.315 0.094 0.087 0.013 MgFc59 0.145 0.378 0.039 0.047 0.033MgFc59/60 0.117 0.273 0.088 0.082 0.012 MgFc31 0.125 0.305 0.040 0.0430.030 MgFc31/60 0.085 0.215 0.139 0.132 0.020 MgFc51 0.135 0.442 0.0600.047 0.062 MgFc51/60 0.098 0.264 0.118 0.106 0.023 MgFc38 0.108 0.2920.034 0.025 0.032 MgFc38/60 0.089 0.232 0.101 0.093 0.021

TABLE 24 KINETIC CHARACTERISTICS OF 4D5 MUTANTS 4D5Mutants 3aV158 3aF1582bfcagl 2aR131Fcagl 2aH131Fcagl MgFc70 0.101 0.250 0.030 0.025 0.025MgFc71 0.074 0.212 0.102 0.094 0.020 MgFc73 0.132 0.306 0.190 — 0.024MgFc74 0.063 0.370 n.b. 0.311 0.166 WT023stable 0.150 0.419 0.071 0.0680.043

7.7 Analysis of Kinetic Parameters of Fc Mutants

Kinetic parameters of binding of chimeric 4D5 antibodies harboring Fcmutants to the two allotypes of FcγRIIIA, FcγRIIA 131H and FcγRIIB wereanalyzed by BIAcore using a method similar to that disclosed in Section7.8 supra. The two allotypes of FcγRIIIA. FcγRIIIA 158V and FcγRIIIA158F, are described in further detail in Section 7.9 supra.

Materials and Methods

Both allotypes of FcγRIIIA used in this assay were soluble monomericproteins, the extracellular region of FcγRIIIA joined to thelinker-AVITAG sequence as described in Section 7.1. The FcγRIIB andFcγRIIA used in this assay were soluble dimeric proteins, i.e. theextracellular domain of FcγRIIB or FcγRIIA fused to the hinge-CH2-CH3domain of human IgG2 as described in Section 7.1 supra.

Details of BIAcore methodology and analysis are found in Section 7.1. Inthis assay, variant Fc regions were cloned into a chimeric 4D5 antibody,which is specific for human epidermal growth factor receptor 2(HER2/neu). The antigen, HER2/neu, was immobilized on one of the flowcells of the sensor chip. The chimeric 4D5 antibodies carrying the Fcmutations were then passed over the surface by 3 minute injections of a300 nM solution at 5 μl/min flow rate. Next, dilution series of thereceptor in HBS-P buffer (10 mM HEPES, 150 mM NaCL, 0.005% SurfactantP20, 3 mMEDTA, pH7.4) were injected onto the surface at 100 μl/min.

Binding curves for two different concentrations of receptor (400 nM and800 nM for both FcγRIIIA V158 and FcγRIIIA 158F; 100 nM and 200 nM forboth FcγRIIA and FcγRIIB) were aligned and responses adjusted to thesame levels of captured antibodies, and reference curves subtracted fromexperimental curves. Association and dissociation phases were separatelyfitted.

Results

Binding of FcγRIIIA, allotype 158 V and 158F, FcγRIIB and FcγRIIA 131Hwere analyzed and resonance responses were normalized at the level ofresponse obtained for a wild type chimeric 4D5 antibody. Kineticparameters for the binding of the FcγRs to the chimeric 4D5 antibodywere obtained by fitting the data at two different FcγR concentrations:400 nM and 800 nM for both FcγRIIIA V158 and FcγRIIIA 158F; 100 nM and200 nM for both FcγRIIA and FcγRIIB.

Table 25 presents the off rate for each of the four receptors analyzedin association with the indicated variant Fc regions.

TABLE 25 Off rate (1/s) of FcγR binding to wild type and mutant chimeric4D5 Ab obtained by BIAcore analysis Chimeric FcγR Receptor 4D5 Fc AminoAcid at Position 3A Region 243 292 300 305 396 3A 158V 158F 2B 2A 131HWild Type F R Y V P 0.186 0.294 0.096 0.073 MgFc0088 L P L I L 0.0160.064 0.058 0.035 MGFc0143 I P L I L 0.017 0.094 0.091 0.049 QuadrupleMGFc0088A L P L L 0.016 0.094 0.075 0.044 MGFc0084 L P I L 0.048 0.1330.278 0.083 MGFc0142 L L I L Triple MGFc0155 L P L 0.029 0.135 0.1550.057 MGFc0074 L P I 0.063 0.37  NB 0.166 MGFc0093 P I L 0.080 0.1970.125 0.190 Double MGFc0162 L P 0.041 0.515 0.900 0.18  MGFc0091 L L0.108 0.330 0.036 0.026 MGFc0070 P I 0.101 0.250 0.030 0.025 SingleSV12/F243L L 0.048 0.255 0.112 0.100 MGFc0161 P 0.067 0.485 0.421 0.117G 0.124 NT 0.384 NT MGFc0092 L 0.211 NT 0.058 0.02  MGFc0089 L 0.1270.306 0.031 0.039

Table 26 presents the results of an duplicate study wherein the K_(off)values of the chimeric 4D5 antibodies were computed relative to wildtype off rates. Relative numbers greater than one indicate a decrease inK_(off) rate. Numbers less than one indicate an increase in K_(off)rate.

TABLE 26 Relative Off-Rate of ch4D5 antibodies obtained by BIAcoreanalysis FcγR Receptor Relative Off Rate (K_(off) WT/K_(off) MUT) 3A 2AChimeric 4D5 Fc Region 3A 158V 158F 2B 131H F243L, R292P, Y300L, V305I,P396L 10.06 8.25 1.38 1.11 F243L, R292P, Y300L 6.69 2.3 0.32 0.65 F243L,R292P, P396L 5.37 3.52 0.32 0.65 F243L, R292P, Y300L, P396L 10.06 5.621.07 0.89 F243L, R292P, V305I 2.56 1.43 nb* 0.23 F243L 4.79 3.44 0.840.57 *nb, no binding

7.8 ADCC Assay of Fc Mutants

Fc mutations identified in Example 7.7 as comprising increased affinityfor FcγIIIA and/or FcγIIA were analyzed for their relative ADCCactivity.

Materials and Methods

Details regarding ADCC assays are found in Section 7.1 and in U.S PatentApplication Publications 2005/0037000 and 2005/0064514, andInternational Patent Application Publication WO 04/063351, each of whichis hereby incorporated by reference in its entirety. In this assay, HT29colon carcinoma cells (ATCC Accession No. HTB-38) loaded with Indium-111were used as targets and effector cells were PBMC that were isolatedfrom donors using a Ficoll gradient. Target cells were opsonized withchimeric 4D5 antibodies comprising the variant Fc regions at finalconcentrations of 2-5000 ng/ml. Opsonized target cells were then addedto effector cells to produce an effector:target ratio of 50:1 andincubated for 18 hours at 37° C., 5% CO₂. After incubation, cells werecentrifuged at ˜220×g for five minutes at 10° C. The level of Indium-111in the supernatant was recorded by a gamma counter.

Results

Chimeric 4D5 antibodies comprising variant Fc regions MGFc88 (F243L,R292P, Y300L, V305I, P396L), MGFc88A (F243L, R292P, Y300L, P396L) andMGFc155 (F243L, R292P, Y300L) were selected based on enhanced affinityfor FcγRIIIA and/or FcγIIA and tested for their ADCC activity. FIGS. 24A& B show that the Fc variants tested exhibit enhanced ADCC activityrelative to wild type antibody at opsonization concentrations above 20ng/ml in a concentration dependent manner. The data indicate that Fcmutants identified as comprising increased affinity for FcγRIIIA arealso likely to exhibit enhanced ADCC activity.

7.9 Fc Mutant Mediated Tumor Growth Control in an In Vivo Tumor Model

Fc mutations identified as comprising enhanced affinity for FcγIIIAand/or FcγIIA were further analyzed for relative efficacy of tumorcontrol using an in vivo tumor model system.

Materials and Methods

Antibodies harboring Fc mutants were tested for anti-tumor activity in amurine xenograft system. Balbc/nude mice are subcutaneously injectedwith 5×10⁶ Daudi cells and subsequently monitored for general signs ofillness, e.g. weight gain/loss and grooming activity. Without treatment,this model system results in 100% mortality with an average survivaltime of approximately 2 weeks post tumor cell inoculation. Treatmentconsists of doses of wild-type antibody or antibody comprising a variantFc region administered at weekly intervals. Animals administered bufferalone at the same intervals serve as a control. Tumor weight iscalculated based on the estimated volume of the subcutaneous tumoraccording to the formula (width²× length)/2.

Results

At weekly intervals, mice inoculated with Daudi cells received wild-typehumanized 2B6 (“h2B6”), humanized 2B6 comprising mutant FcMG0088 (F243L,R292P, Y300L, V305I P396L) (“h2B6 0088”) or buffer alone. Wild-type andFc mutant h2B6 antibody showed similar levels of tumor suppression atthe highest dose schedule tested, weekly doses of 25 μg (FIGS. 25A andB). However, significant differences in antibody efficacy were observedwhen dosages were reduced. 100 and 10 fold reduction in wild-type h2B6dosages provided no greater tumor control than administration of bufferalone (FIG. 42A). In contrast, h2B6 0088 provided significant protectionat weekly doses of 2.5 μg and at least limited protection at weeklydoses of 0.25 μg (FIG. 25B).

The protection conferred by even the lowest dose of Fc mutant antibodywas confirmed in survival comparisons. At 11 weeks, 4 out of 7 miceremained alive in the group treated with 0.25 μg doses of h2B6 0088compared to only 1 out of 7 in the group treated with the same dose ofwild-type h2B6 (FIGS. 26A & B)

7.10 Fc Mutant Mediated Tumor Growth Control in a Human Fc ReceptorExpressing Transgenic Mouse Tumor Model

Fc mutations identified as comprising enhanced affinity for FcγIIAand/or FcγIIA were further analyzed for relative efficacy of tumorcontrol using an in vivo xenograft human Fc receptor transgenic mousetumor model system.

Materials and Methods

Humanized antibodies against human CD32B (h2B6) or HER2/neu (h4D5)harboring Fc mutations were tested for anti-tumor activity in a murinexenograft system, in which mouse FcγIIIA (CD16) was replaced with itshuman orthologue, CD16A (huCD16A). Immunodeficient mice were injectedwith 5×10⁶ tumor cells and subsequently monitored for general signs ofillness, e.g. weight gain/loss and grooming activity. Treatment consistsof doses of wild-type antibody or antibody comprising a variant Fcregion administered at daily or weekly intervals (as stated). Animalsadministered buffer alone or antibody comprising mutation N297Q (whichabrogates binding to any FcγR) at the same intervals serve as a control.Tumor weight was calculated based on the estimated volume of thesubcutaneous tumor according to the formula (width²× length)/2.

Results

h2B6: Humanized Anti-CD32B and Fc Variants

At weekly intervals beginning two weeks subsequent to tumor injection,RAG1−/− C57BI/6 mice subcutaneously injected with Raji cells(CD32B-expressing tumor cells) received wild-type humanized 2B6 (“h2B6”;Rituxan), humanized 2B6 comprising mutant FcMG0088 (F243L, R292P, Y300L,V305I P396L) (“h2B6 0088”; “FcMg88”, or MGA321) or buffer alone.Wild-type and Fc mutant h2B6 antibody showed similar levels of tumorgrowth suppression at weekly doses of 250 μg and 25 μg (FIG. 27).However, significant differences in antibody efficacy were observed whenthe dosage was reduced to 2.5 μg: At this dosage, wild-type h2B6provided limited tumor growth control compared with administration ofbuffer alone; in contrast, the 2.5 μg dosage of h2B6 0088 delayed tumorprogression by as much as one week (FIG. 27). In another experiment, theefficacy of the lower dosages of Fc-optimized MGA321 tested wereequivalent to controls (PBS or Rituxin at equivalent dosages); however,administration of the highest dosage (250 μg) of Fc-optimized h2B6antibody provided significant tumor growth control compared withwild-type h2B6- and buffer alone-treated mice (FIGS. 28A-2B).

The protection conferred by the Fc mutant antibody was confirmed insurvival comparisons. Nude (FoxN1) mice were intraperitoneally injectedwith EL4-CD32B cells and then treated on days 0, 1, 2, 3, and 6 with IPadministered humanized 2B6 1.3 or humanized 2B6 1.3 comprising mutant31/60 (P247L. D270E, N421K) (“h2B6 1.3 3160”). At 14 weeks, at least 90%of mice treated with h2B6 1.3 3160 survived compared with 55% or less inthe group treated with the same dose of wild-type h2B6 1.3 (FIG. 29 andFIG. 30).

In a further survival experiments in the same system, mice were treatedwith IP administered humanized 2B6 3.5, humanized 2B6 3.5 comprisingmutant FcMG0088 (F243L, R292P, Y300L, V305I, P396L) (“h2B6 3.5 0088”),humanized 2B6 3.5 N297Q (negative control) or buffer alone. At 14 weeks,all mice treated with h2B6 3.5 0088 survived compared with 30% in thegroup treated with the same dose of wild-type h2B6 3.5 and <20% in thegroups treated with the N297Q mutant or PBS (FIG. 31A; treatment on day0, 1, 2, 3); the same result was achieved for a dose as low as 4 μg/gbody weight (FIG. 31B; treatment on day 0, 1, 2, 3, 4)

The protective effects of antibodies comprising variant Fc regions wasfurther tested in transgenic mice carrying human CD32A in addition tothe mCD16−/− huCD16A+ genotype using the EL4-CD32B model describedsupra. Treatment with wild-type h2B6 or negative control, h2B6 3.5N297Q, resulted in only 20% at 100 days post tumor inoculation;treatment with Fc optimized antibody, h2B6 3.5 0088, increased survivalby 10%, with 30% survival at 100 days post inoculation (FIG. 32).

The effect of the expression of hCD16A and/or hCD32A in the transgenicmurine EL4-CD32B tumor model on treatment with h2B6 antibodies wasfurther investigated using nude (FoxN1) mice positive for hCD16A,positive for both hCD16A and hCD32A, or positive for hCD32A, each on amCD16−/− background. Transgenic were intraperitoneally injected withEL4-CD32B cells and then treated at day 0, 1, 2, and 3 with wild-typehumanized 2B6 3.5 (“h2B6 3.5”), humanized 2B6 3.5 comprising mutantFcMG0088 (F243L, R292P, Y300L, V305I, P396L) (“h2B6 3.5 88”), h2B6 N297Q(“h2B6 3.5 N297Q;” negative control) or buffer alone. At 100 days posttumor inoculation, all CD16A positive mice treated with h2B6 3.5 88survived compared with 50% or less survival in the groups treated withthe same dose of wild-type h2B6 3.5 or controls (FIG. 33A). In miceharboring human CD32A in addition to the mCD16−/− huCD16A+ genotype,survival was not more than 25% at 100 days post inoculation regardlessof treatment (FIG. 33B). In mice expressing hCD32A but not hCD16, onlymice in the Fc-optimized treatment group, treated with h2B6 3.5 88,survived longer than 1 month, with 25% survival at 100 days postinoculation (FIG. 33C).

The effect of different time courses of treatment with Fc-optimizedantibodies (h2B6 0088; “MGA321”) on mouse survival was alsoinvestigated. Intraperitoneal injection of mice with MGA321 immediatelyafter tumor injection conferred survival on 75%-100% of mice, either ata single or multiple doses over consecutive days or weeks (FIG. 34).When MGA32I was first administered a day or later subsequent to tumorintroduction, it conferred a maximum of 40% survival, even whenadministered in multiple doses over days or weeks (FIG. 34).

ch4D5: Chimeric anti-HER2/neu and Fc variants.

A HER2/neu positive tumor model was established by IP injection ofmSKOV3 cells (HER2/neu-expressing ovarian tumor cells) into mCD16knockout nude (FoxN1) mice that also carried, and expressed, human CD16Aor both human CD16A and human CD32A. At 8 weekly intervals, starting attime 0, inoculated mice were subcutaneously injected with “wild type”chimeric antibody against human HER2/neu (ch4D5), ch4D5 comprisingFcMG0088 (F243L, R292P, Y300L, V305I P396L) (“ch4D5 0088”), ch4D5comprising N297Q (aglycosylated negative control; “ch4D5 N297Q”), orbuffer alone. Treatment with ch4D5 0088 suppressed tumor growth for theentire course of the experiment (10 weeks) in transgenic mice positivefor human CD16A on the mCD16−/− background or in transgenic miceharboring human CD32A in addition to the mCD16−/− huCD16A+ genotype(FIG. 35A or 35B, respectively).

The protection conferred by the Fc mutant ch4D5 antibody was confirmedin survival comparisons. Knockout mCD16 (mCD16−/−) nude (N/N) micetransgenic for human CD16A were intraperitoneally injected with mSKOV3cells and then treated with wild-type chimeric 4D5 (“ch4D5”), ch4D5comprising FcMG0088 (F243L, R292P, Y300L, V305I P396L), ch4D5 N297Q(negative control), or buffer alone. Tumor inoculated mice received sixdoses beginning at day 0 (day of tumor inoculation) of 100 μg or 1 μg ofthe antibody delivered IP (FIGS. 36A and 36B, respectively). At 14weeks, ˜60% of mice treated with 100 μg ch4D5 0088 survived comparedwith ˜40% in the group treated with the same dose of wild-type ch4D5 and<10% of mice treated with ch4D5 N297Q or buffer alone (FIG. 36A). Atdoses of 1 μg, the overall duration of survival of mice treated withwild-type or Fc-optimized antibodies was reduced relative to thosetreated with 100 μg. However, at six weeks, more than 80% of ch4D50088-treated mice were still alive, compared with ˜10% of mice thatreceived the other treatments (FIG. 36B), confirming that a moderatedose of Fc-optimized ch4D5 confers a significant improvement over thewild-type antibody in enhancing viability.

Other Fc variants of ch4D5 were tested in similar survival experiments.

A chimeric 4D5 comprising mutant MGFc0155 (F243L, R292P, Y300L) (“ch4D50155”) or mutant MCFc3160 (P247L, D270E, N421K) (“ch4D5 3160”) weretested alongside wild-type ch4D5, ch4D5 N297Q, and ch4D5 0088 asdescribed supra. Mice received eight weekly intraperitoneal treatments,starting at day 0 (tumor inoculation), with 100 μg antibody. In micereceiving doses of 100 μg, 100% of the group treated with ch4D5 0155were alive at 130 days post inoculation, compared with ˜85% of thosetreated with ch4D5 0088 and 50% of those treated with ch4D5 3160. Incontrast, only approximately 30% of mice given wild-type ch4D5 werestill alive at day 130. All mice treated with buffer alone or ch4D5N297Q died within 14 weeks and 10 weeks, respectively, after tumorinjection (FIG. 37A). At a 10-fold lower dosage, Fc-optimized antibodieswere less efficacious in enhancing survival compared with wild-typech4D5. While ch4D5 0155 was most effective at enhancing survival atearlier time points, at 18 weeks, ˜60% of mice in the group treated withch4D5 0155 or ch4D5 0088 survived, compared with 50% ofwild-type-treated and 25% of ch 4D5 3160-treated (FIG. 37B).

In another set of survival experiments, Knockout mCD16 (mCD16−/−) nude(N/N) mice transgenic for human CD16A or transgenic for both human CD16A(“hCD16”) and human CD32A (“hCD32A”) were intraperitoneally injectedwith mSKOV3 cells and then, starting at day 0, given eight weeklytreatments with wild-type ch4D5, ch4D5 0088, ch4D5 N297Q or bufferalone. At seven weeks post-tumor injection, all ch4D5 0088-treated andhCD16 positive mice were alive, whereas only ˜30% of Ch4D5 N297Q and˜10% of buffer-treated hCD16 positive mice survived (FIG. 38A). In nudemice harboring human CD32A in addition to the mCD16−/− huCD16A+genotype, 50% or the mice treated with ch4D5 0088 were alive after eightweeks, compared with those that received ch4D5 N297Q (15% alive at eightweeks) or buffer alone (all dead before six weeks) (FIG. 38B).

7.11 Fc Variants Exhibiting Altered Ratios of Affinities

Immunoglobulins whose Fc regions had been mutated in the mannerdescribed above are screened for Fc variants having altered Ratios ofAffinities to FcγRIII and FcγRII by assessing the K_(off) of thevariants and their wild-type immunoglobulin progenitors. Testing is doneusing both the V158 and F158 isotypes of FcγRIII, and against FcγRIIBand FcγRIIAH 131. The results are summarized in Table 27.

TABLE 27 COMPARISON OF K_(OFF) OF FC MUTANTS TO WILD TYPE ANTIBODY(K_(OFF) WT/K_(OFF) MUTANT) Ratio of Affinities CD16A CD16A CD32ACD16A/CD32B Fc sequence V158 F158 CD32B H131 V158 F158 WT 1.00 1.00 1.001.00 1.00 1.00 F243L 4.79 3.44 0.84 0.57 5.70 4.10 D270E 1.25 1.48 0.392.24 3.21 3.79 R292P 2.90 0.64 0.25 0.53 11.60 2.56 R292G 1.54 — 0.250.14 6.2 — Y300L 1.01 1.17 1.18 1.86 0.86 0.99 P396L 1.27 1.73 2.58 1.630.49 0.67 P396L D270E 1.38 1.65 0.89 2.44 1.55 1.85 P396L F243L 1.491.60 2.22 1.50 0.67 0.72 P247L N421K 1.29 1.73 2.00 1.30 0.65 0.87 P247LN421K D270E 1.89 2.46 0.58 1.95 3.26 4.24 P247L N421K F243L 1.89 1.710.17 0.39 11.12 10.06 P247L N421K D270E F243L 2.30 3.45 0.32 0.98 7.1910.78 P247L N421K D270E Y300L 2.44 1.16 0.8  1.84 3.05 1.45 R255L P396L1.09 1.39 2.22 1.34 0.49 0.63 R255L P396L D270E 1.34 1.65 0.87 3.00 1.541.90 R255L P396L D270E F243L 1.75 1.64 0.38 1.44 4.61 4.32 R255L P396LD270E R292G 1.39 1.30 0.65 1.05 2.14 2.00 R255L P396L D270E Y300L 1.521.74 0.87 2.60 1.75 2.00 K392T P396L 1.49 1.81 2.35 1.22 0.63 0.77 K392TP396L D270E 1.81 2.28 0.79 1.86 2.29 2.89 K392T P396L D270E F243L 3.162.44 0.44 1.70 7.18 5.55 Q419H P396L 1.19 1.19 1.33 0.63 0.89 0.89 Q419HP396L D270E 1.64 2.00 0.68 1.70 2.41 2.94 Q419H P396L D270E F243L 1.461.15 0.26 1.11 5.62 4.42 R292P F243L 4.73 0.6 0.12 0.34 39.4 5.00 R292PF243L 4 1.67 0.16 0.52 25 10.44 R292P V284M K370N 1.14 1.37 0.37 1.793.1 3.7 R292P V305I 1.59 2.11 2.67 1.56 0.60 0.79 R292P V305I 1.32 1.280.37 0.75 3.6 3.46 R292P V305I F243L 2.56 1.43 ND 0.23 >25 >25 R292PV305I F243L P396L 5.37 2.53 0.40 0.78 13.43 6.33 R292P V305I F243L P396LY300L 10.06 8.25 1.38 1.11 7.29 5.98 R292P V305I F243I P396L Y300L 10.93.12 1.05 1.49 10.4 2.97 R292P F243L P396L Y300L 10.06 5.62 1.07 0.899.40 5.25 R292P F243L P300L 6.69 2.3 0.32 0.65 20.9 7.19 R292P V305IP396L 1.85 1.90 0.92 1.50 2.01 2.07 R292P V305I P396L F243L 5.37 2.530.40 0.78 13.43 6.33 G316D R416G D270E 2.18 2.49 0.78 1.95 2.79 3.19G316D R416G D270E F243L 1.50 1.34 0.20 1.22 7.50 6.70 G316D R416G D270EP396L 1.22 0.94 1.07 0.95 1.14 0.88 Fc mutants analyzed in this studyare shown in the left hand column. The dissociation rate constants forbinding of the Fc to the different FcR were determined by Biacoreanalysis. ND = no detectable binding

The results show that the methods of the present invention are capableof producing both molecules that possess Fc regions having a Ratio ofAffinities greater than wild-type (i.e., >1) as well as molecules thatpossess Fc regions having a Ratio of Affinities less than wild-type(i.e., <1). An analysis of the Fc variants shows that the variantFc-containing molecules fall into various classes, as shown in Table 28.

TABLE 28 Ratio of Affinities CD16A/ CD16A CD16A CD32B Fc sequence V158F158 CD32B V158 F158 Ratio of Affinities >1 Class I: Increased Bindingto CD16; Decreased Binding to CD32B F243L 4.79 3.44 0.84 5.70 4.10 D270E1.25 1.48 0.39 3.21 3.79 R292P 2.90 0.25 11.60 R292G 1.54 0.25 6.2 P396LD270E 1.38 1.65 0.89 1.55 1.85 P247L N421K D270E 1.89 2.46 0.58 3.264.24 P247L N421K F243L 1.89 1.71 0.17 11.12 10.06 P247L N421K D270E 2.303.45 0.32 7.19 10.78 F243L R255L P396L D270E 1.34 1.65 0.87 1.54 1.90R255L P396L D270E 1.75 1.64 0.38 4.61 4.32 F243L R255L P396L D270E 1.391.30 0.65 2.14 2.00 R292G R255L P396L D270E 1.52 1.74 0.87 1.75 2.00Y300L K392T P396L D270E 1.81 2.28 0.79 2.29 2.89 K392T P396L D270E 3.162.44 0.44 7.18 5.55 F243L Q419H P396L D270E 1.64 2.00 0.68 2.41 2.94Q419H P396L D270E 1.46 1.15 0.26 5.62 4.42 F243L R292P F243L 4.73 0.1239.4 R292P F243L 4 1.67 0.16 25 10.44 R292P V284M K370N 1.14 1.37 0.373.1 3.7 R292P V305I 1.32 1.28 0.37 3.6 3.46 R292P V305I F243L 2.56 1.43ND >25 >25 R292P V305I F243L 5.37 2.53 0.40 13.43 6.33 P396L R292P F243LP300L 6.69 2.3 0.32 20.9 7.19 R292P V305I P396L 5.37 2.53 0.40 13.436.33 F243L G316D R416G D270E 2.18 2.49 0.78 2.79 3.19 G316D R416G D270E1.50 1.34 0.20 7.50 6.70 F243L G316D R416G D270E 1.22 1.07 1.14 P396LClass II: Decreased Binding to CD16; Greatly Decreased Binding to CD32BR292P 0.64 0.25 2.56 R292P F243L 0.6 0.12 5.00 Class III: IncreasedBinding to CD16; Unchanged Binding to CD32B R292P V305I F243I 10.9 3.121.05 10.4 2.97 P396L Y300L R292P F243L P396L 10.06 5.62 1.07 9.40 5.25Y300L R292P V305I P396L 1.85 1.90 0.92 2.01 2.07 Class IV: GreatlyIncreased Binding to CD16; Increased Binding to CD32B R292P V305I F243L10.06 8.25 1.38 7.29 5.98 P396L Y300L G316D R416G D270E 1.22 1.07 1.14P396L Ratio of Affinities <1 Class V: Unchanged Binding to CD16;Increased Binding to CD32B Y300L 1.01 1.18 0.99 R255L P396L 1.09 2.220.49 Class VI: Increased Binding to CD16; Greatly Increased Binding toCD32B P396L 1.27 1.73 2.58 0.49 0.67 R255L P396L 1.39 2.22 0.63 P396LF243L 1.49 1.60 2.22 0.67 0.72 P247L N421K 1.29 1.73 2.00 0.65 0.87R255L P396L 1.39 2.22 0.49 0.63 K392T P396L 1.49 1.81 2.35 0.63 0.77Q419H P396L 1.19 1.19 1.33 0.89 0.89 R292P V305I 1.59 2.11 2.67 0.600.79 Class VII: Decreased Binding to CD16; Increased/Unchanged Bindingto CD32B G316D R416G D270E 0.94 1.07 0.88 P396L

7.12 Predictive Efficacy of Ratios of Affinities

Fc domains of Fc variants that exhibited improved Ratios of Affinitiesin the context of the spectrum of murine FcγRs are evaluated todetermine their in vivo efficacy. For such purpose, the Fc domains wereincorporated into a prototype therapeutic antibody and tested inxenograft mouse models of B-cell lymphoma and in tumor models inFcγRIII-knock-out mice that express the low-binding allele of humanCD16A. The impact of Fc engineering on tumor clearance was investigatedby using WT or human FcγR-transgenic mice. Hu2B6 was used as the modelmAb, since this antibody does not induce complement lysis or apoptosis,but inhibits tumor growth in mice by mechanisms that are exquisitely Fcγdependent (Rankin, C. T. et al. 2006 Blood 108:2384-2391). Because hu2B6does not cross-react with murine (m) FcγRII or other endogenous murineproteins, there is no antibody target other than the implantedCD32B-positive tumor cells in this model (Rankin, C. T. et al. 2006Blood 108:2384-2391). Furthermore, hu2B6 completely blocks human CD32B,thus eliminating binding of the hu2B6 Fc region to the target cells as aconfounding factor. Fc variants 088, 3160, 5660, 3860 and 0071 wereselected for this purpose.

Fc variants 088 and 3160 are tested for treatment of B cell tumors in16A tg mice. Fc variants 088, 3160, 5660, 3860 and 0071 were tested fortreatment of B cell tumors in Balb/c mice.

Mouse Tumor Models

Xenograft Models:

Female athymic Balb/c nude (nu/nu) mice, 8-10 weeks old, are purchasedfrom Taconic. Daudi cells (5×10⁶ per mouse) are suspended inPBS+Matrigel and injected subcutaneously into the right flank of Balb/cnude mice. Tumor development is monitored twice per week, usingcalipers, and tumor weight is estimated by the following formula: tumorweight=(length× width²)/2. Intraperitoneal (IP) injections of antibodiesat various concentrations (1 μg/g, or 0.1 μg/g) are performed weekly for6 weeks, starting at day 0.

EL4/CD32B Model:

Male and female athymic mFcγRIII^(−/−), hCD16A⁺ nude mice, are bred inMacroGenics, Inc. animal facility. EL4/CD32B cells (1×10⁴ per mouse) aresuspended in PBS and injected IP at day 0. IP injections of antibodies(10 μg/g or 4 μg/g) are performed on Days 0, 1, 2, and 3. Mouse bodyweight is measured twice a week. Mice showing excessive body weight gainas well as signs of ascites tumors are sacrificed by CO₂ asphyxiation.Survival was recorded accordingly. Data is analyzed using PRISM(Graphpad Software, San Diego Calif.) for calculation of standarddeviation (ADCC, tumor model) and statistical significance using T-testsand log rank analysis (tumor models).

For a complete mechanistic interpretation of the data, the bindingprofiles of the engineered Fcγ to the mouse (m) FcγRs were fullycharacterized. Of the mFcγR, mFcγRII is structurally and functionallyhomologous to human CD32B, while mFcγRIII and mFcγRIV are receptorsfunctionally related to human activating FcγRs expressed on NK cells andmonocyte/macrophages, respectively (Nimmerjahn, F. et al. 2005 Science310:1510-1512; Nimmerjahn, F. 2005 Immunity 23:41-51). Since the ratioof Fcγ-binding to activating and inhibitory FcγRs is shown to beimportant in determining antibody-dependent outcomes in vivo(Nimmerjahn, F. et al. 2005 Science 310:1510-1512; Nimmerjahn, F. 2005Immunity 23:41-51), mutants are selected based on their binding profiles(Table 29) using human FcRs. Data in Table 29 is expressed as foldchanges relative to wild-type affinities.

TABLE 29 MGFc CD16/ No. Fc Mutant CD16A^(H) CD16A^(L) CD32A^(H) CD32BCD32B 0193 F243L +3.8 +2.4 −0.8 −0.2 5.7 0089 P396L +0.3 +0.7 +0.6 +1.60.5 3160 P247L D270E N421K +0.9 +1.5 +1.0 −0.7 3.3 5560 R255L D270E:P396L +0.3 +0.7 +2.0 −0.1 1.5 3860 D270E K392T P396L +0.8 +1.3 +0.9 −0.32.3 0074 F243L R292P v3051 +1.6 +0.4 −3.3 −13.3 36.6 0071 D270E G316DR416G +0.4 +0.1 +0.4 −1.7 3.8 0155 F243L R292P Y300L +6.4 +3.6 0.0 −0.712.3 0031 P247L N421K +0.3 +0.7 +0.3 +1.0 0.6 0161 R292P +1.4 +0.6 −0.5−2.7 8.7 0162 F243L R292P +3.0 +0.7 −0.9 −5.3 24.7 0170 F243L R292PP396L +5.3 +2.4 +0.4 −1.6 16.0 0092 Y300L 0.0 +0.2 +1.9 +0.2 0.9 0088F243L R292F Y300L V305I P396L +9.1 +7.3 +2:2 +0.4 7.3 0084 F243L R292PV305I P396L −3.0 +1.3 −0.3 −1.6 10.6 Controls Wild-Type 0.0 0.0 0.0 0.01.0 AAA* E333A K334A S298A +0.6 +0.1 −3.3 −2.5 5.7 XmAb** S239D I332E330L +13.9 +8.1 0.0 +0.74 8.5 Shading indicates Fc versions tested inmouse tumor models. *see, Presta, L., U.S. Pat. No. 6,737,056; Shieldset al. 2002, J Biol Chem 277: 26733-26740 **see, Lazar, G. A. et al.Proc. Natl. Acad. Sci. (USA) 103: 4005-4010 (2006)

Mutants MGFc3160 (P247E/D270E/N421K) and MGFc0088 showed increasedbinding to mFcγRIII and mFcγRIV, respectively. MGFc3160, however, showedconcomitantly increased mFcγRII binding, while MFc0088 exhibited a morefavorable activating-to-inhibitory profile, with no increase in bindingto the inhibitory receptor. Mutant MGFc0071 (D270E/G316D/R416G)demonstrated WT Fcγ-level binding to all mFcγRs (Table 29) and wasincluded as a control.

Sub-cutaneous inoculation of Daudi cells in nude mice produceslocalized, progressively expanding tumors whose growth is significantlyreduced by weekly intra-peritoneally (i.p.) injections of 1 μg/g WThu2B6 (FIG. 39A). Weekly 0.1 μg/g doses, however, are ineffective. Atthe higher dose, treatment with hu2B6-MGFc3160 is indistinguishable fromthat with WT hu2B6, but a modest improvement over WT hu2B6 is detectableat the lower dose. hu2B6-MGFc0088, however, resulted in a significantreduction in tumor growth at all doses tested, consistent with itsenhanced mFcγRIV binding and highly favorable activating-to-inhibitoryratio due to no corresponding increase in mFcγRII binding.Hu2B6-MGFc0071, which showed WT level binding to all mFcγRs, behavedsimilarly to WT hu2B6 (FIG. 39B).

Enhanced Tumor Clearance in Human CD16A Transgenic Mice

The activity of Fc-engineered mAbs is further analyzed inmFcγRIII-knockout mice expressing the transgene for the low-affinityallele of human CD16A (Li, M. et al. 1996 J. Exp. Med. 183:1259-1263).In these mice, human CD16A-158^(phe) is expressed by NK cells andmononuclear phagocytes, similarly to its cell-type specific expressionin humans (Perussia, B. et al. Eur. J. Immunol. 21:425-429). The murinecell line, EL4 (Gorer, P. A. (1950) Brit. J. Canc. 4(4):372-379), wastransduced with human CD32B and used in place of Daudi cells, whosetumor take was poor in these transgenic mice. Knock-out transgenic mice,mFcγRIII^(−/−)/CD16A-158^(phe+), injected i.p. with CD32B-EL4 cells,died eight weeks after inoculation. Treatment with hu2B6-WT rescued 40%of the animals, whereas 90% survived after receiving hu2B6-MGFc3160(FIG. 39C). In a separate experiment, a regimen of hu2B6-MGFc0088 thatdid not prevent tumor growth as a WT Fcγ showed 100% mouse survival forthe duration of the experiment (FIG. 39D). Therefore, the potency ofhu2B6-MGFc3160 and hu2B6-MGFc0088 in vivo ranks consistently with theirrelative improvement in binding to FcγRs expressed in the mice (Table29).

Thus, the Ratio of Affinities of an Fc variant is found to be predictiveof the in vivo efficacy of molecules comprising the Fc variant region.Both hu2B6-MGFc3160 and hu2B6-MGFc0088 showed enhanced inhibition oftumor cell growth compared to WT mAb. Since MGFc3160 showed an isolatedenhancement in mFcγRIII binding in the absence of improved mFcγRIVinteraction, the increased activity could be attributable to both NKcells and mononuclear phagocytes. On the contrary, the properties ofMGFc0088, an Fcγ domain with substantially increased affinity to mFcγRIVbut mFcγRIII binding properties similar to those of WT, were consistentwith the notion that mononuclear phagocytes are the critical cells forimproved tumor elimination. In the huFcγR transgenic mouse, thesubstitution of mFcγRIII with huCD16A-158^(phe) on both NK cells andmonocytes resulted in an increase in the activating-to-inhibitorybinding ratio for MGFc0088. Again, the greater increase in mousesurvival with hu2B6-MGFc0088 correlated with its increased affinity forthe second activating receptor, mFcγRIV, expressed bymonocytes/macrophages (Nimmerjahn, F. et al. 2005 Immunity 23:41-51).The ability of Fc variants to bind mixed human/murine FcγRIII and FcγRIIreceptors was determined. The results (Table 30) indicate that thevariants bind to chimeric receptors with substantial equivalence.

TABLE 30 hCD16 hCD16 hCD16 No. Fc Mutant mCD32 mCD32 mCD32 0071 D270EG316D R416G 1.0 1.1 3160 P247L D270E N421K 1.7 0.6 2.2 5560 R255L D270EP396L 0.7 1.5 3860 D270E K392T P396L 0.7 1.5 0088 F243L R292F Y300LV305I P396L 10.7 17.3 1.4The binding of selected Fc variants to Fc receptors, and their Ratios ofAffinities is shown in Table 31.

TABLE 31 32A- 16A- 16A- 3aV 3aF 2bFcagl 2aRFcagl 2aHFcagl 131H:32B158V:32B 158F:32B wt 1 1 1 1 1 1 1 1 L234F, 1.7 1.6 0.2 0.2 0.3 1.5 8.58 F243L, R292P, Y300L L235I, 2.6 3.3 0.2 0.1 0.5 2.5 13 16.5 F243L,R292P, Y300L L235Q, 1.8 1.3 n.d. n.d. 0.2 n.d. n.d. n.d. F243L, R292P,Y300L L235V, 6.1 4.8 0.4 0.4 1.3 3.3 15 12 F243L, R292P, Y300L, P396LL235P, 5.4 2.5 0.2 0.2 0.7 3.5 27 12.5 F243L, R292P, Y300L, P396L

7.13 Fc Variants of HER2-Reactive Antibodies Exhibiting DiminishedFucosylation

As discussed above, one aspect of the present invention relates to therecognition that variations in the Fc region of an antibody caninterfere with the cellular glycosylation mechanism and thereby yieldantibodies that exhibit a decreased extent of glycosylation (and inparticular, fucosylation). In order to demonstrate the effect of suchvariations in the Fc region on the capacity of cells to fucosylate theanti-Her2 antibodies of the present invention, a series of variants ofthe anti-Her2/neu antibody ch45D4-FcMT2 were constructed (Table 32).

Antibody ch4D5-FcMT2 has a light chain having an amino acid sequence ofSEQ ID NO: 45, and a heavy chain having an amino acid sequence of SEQ IDNO: 46, ch4D5-FcMT2 has an N65S substitution on the light chain, whichresults in a de-glycosylated light chain, and L235V, F243L, R292P,Y300L, and P396L substitutions on the heavy chain (all numberedaccording to Kabat). This antibody binds to CD16A (FcγRIII-A), andbinding to CD16-158^(Phe) is enhanced in a proportionally greaterfashion than binding to CD16-158^(Val), but exhibits reduced binding toCD32B (FcγRII-B). Polynucleotides encoding the ch4D5-FcMT2 light andheavy chains are provided in SEQ ID NO:47 and 48, respectively.

Table 32 shows the ratio of wild-type K_(off)/mutant K_(off) forantibodies having the indicated Fc variations. Results for antibodych45D4-FcMT2 are shown in boldface in the shaded row of Table 32 (n.b.,no binding; n.d., no detectable binding).

TABLE 32 Wild-Type K_(off)/Mutant K_(off) CD16A CD16A CD32A CD32A AminoAcid Changes (158V) (158F) CD32B (131H) (131R) L235V F243L R292P Y300L2.3 2 n.d. 0.5 n.d. L235R F243L R292P Y300L 1.2 0.3 n.d. n.d. n.d. L235WF243L R292P Y300L 1.4 0.9 0.5 1.4 0.6 L235G F243L R292P Y300L 0.8 0.2n.d. n.d. n.d. L235P F243L R292P Y300L 2.6 1.1 n.d. 0.2 n.d. L235N F243LR292P Y300L 1.6 0.7 n.d. 0.2 n.d. L235T F243L R292P Y300L 2.2 1.5 0.10.4 0.1 L235S F243L R292P Y300L 1.7 1 0.1 0.3 0.1 L235H F243L R292PY300L 2 1.1 0.8 1.4 0.8 L235E F243L R292P Y300L 1.8 1 0.2 0.4 0.2 L235FF243L R292P Y300L 1.8 1 0.4 1.3 0.4 L235Y F243L R292P Y300L 1.9 1.1 0.41.4 0.4 L235K F243L R292P Y300L 0.7 0.5 n.b n.b n.b. L235V F243L R292PY300L P396L 6.1 4.8 0.4 1.3 0.4 L235P F243L R292P Y300L P396L 5.4 2.50.2 0.7 0.2 L235C F243L R292P Y300L n.b. n.b. n.b. n.b. n.d L235A F243LR292P Y300L 2.4 1.3 n.d. 0.4 0.2 L235D F243L R292P Y300L 1.9 1.1 0.3 0.40.3 L234F F243L R292P Y300L 1.7 1.6 0.2 0.3 0.2 L235M F243L R292P Y300L2.3 1.3 0.2 0.3 0.3 L235I F243L R292P Y300L 2.6 3.3 0.2 0.5 0.1 L235QF243L R292P Y300L 1.8 1.3 n.d. 0.2 n.d. L235T F243L R292P Y300L P396L4.2 2.8 0.3 0.9 0.3 L235A F243L R292P Y300L P396L 4.4 2.6 0.4 1.1 0.4L234F F243L R292P Y300L P396L 3.5 2.9 0.3 1.2 0.4 L235I F243L R292PY300L P396L 4.5 4.3 0.5 1.6 0.6 L235Q F243L R292P Y300L P396L 3.9 2 0.10.7 0.2 L234F L235V F243L R292P Y300L P396L 1.9 2 n.d. 0.5 0.2 L234FL235I F243L R292P Y300L 1.2 1 n.d. 0.3 n.d. L234F L235I F243L R292PY300L P396L 1.9 2.3 n.d. 0.9 0.2

For further comparison, Table 3 shows the ratio of wild-typeK_(off)/mutant K_(off) for Ch4D5 anti-CD20 (rituximab) antibodies havingthe indicated Fc variations (see. Stavenhagen, J. B. et al. (2007) “FcOptimization of Therapeutic Antibodies Enhances Their Ability to KillTumor Cells In vitro and Controls Tumor Expansion In vivo viaLow-Affinity Activating Fcγ Receptors,” Cancer Res. 67(18):8882-8890).

As is apparent from Tables 3 and 32, antibodies having an Fc variantcontaining a substitution at position L234 or L235, particularly incombination with a substitution at any one or more of positions F243,8292, Y300, V305, and P396, exhibit altered Fc binding to FcγRIII (e.g.,improved binding to the activating receptors (e.g., CD16A, CD32A) andreduced binding to CD32B.

The glycosylation of antibody ch45D4-FcMT2 was investigated using MAbNeutral Monosaccharide Analysis (3 Hour Hydrolysis)” (to investigateGLY-2-2-4 glycosylation) and by “N-Linked Oligosaccharide Profiling ofNeutral Oligosaccharides using an Aqueous Chromatographic Separation”(to investigate GLY-12-3-2 glycosylation). The results of thisinvestigation are shown in FIG. 40, Panels A-D. FIG. 40, Panel A showsthe assignment of N-linked oligosaccharides as determined using anantibody reference panel. As is apparent from FIG. 40, the glycan peaksobserved for antibody ch45D4-FcMT2 (Panels B and C) do not correspond toany of the glycans shown in Panel A. The identity of the glycans isreadily determined by conducting a chromatographic analysis of theantibody ch45D4-FcMT2 glycans in the presence of an added known glycan,and identifying the known glycan that causes an increase in the peaksize of any of the peaks shown in FIG. 40, Panels B and C.

The monosaccharide computational analysis of the antibodies is shown inTable 33 shows the pmol/injection and mol/mol ofmonosaccharides/antibody for the monosacchrides fucose (Fuc),N-acetylated galactose (GALNAc), N-acetylated glucose (GlcNAc),galactose (Gal) and mannose (Man). In Table 9, BLQ denotes that theconcentration of monosaccharide was below the limit of quantification.The very high levels of mannose identified in the analysis indicatesthat elevated extents of manosylation enhance the therapeutic efficacy(as measured, for example by the ratio of mutant K_(off)/wild-typeK_(off)) of antibodies having the variant Fc regions of the presentinvention.

TABLE 33 Monosaccharide Compositional Analysis ch45D4- FcMT2 Fuc GalNAcGlcNAc Gal Man Sample 1 pmol/injection BLQ* BLQ 165.8 BLQ 1033.0 BLQ BLQ176.8 BLQ 1014.7 BLQ BLQ 205.9 BLQ 1135.8 mol/mol ≦0.2 ≦0.3 1.8 ≦0.510.5 Sample 2 pmol/injection BLQ BLQ 77.0 BLQ 698.7 BLQ BLQ 104.4 BLQ836.4 BLQ BLQ 127.7 BLQ 924.4 mol/mol ≦0.2 ≦0.3 1.0 ≦0.5 8.1

7.14 Sites of Fc Variation Associated with Diminished Fucosylation

In order to assess the effect of variations at positions 234, 235, 243,292, 300 and 396, alone or in combination with one another, on thefucosylation of IgG Fc regions, a series of Fc variants was preparedusing the IgG ch4D5 (chimeric anti-Her2/neu), h2B6 3.5 (anti-FcγRIIB;see US 2008/0044417), ch2.4G2 (rat anti-mouse FcγRII-III; Kurlander etal. (1984) “The Blockade Of Fc Receptor-Mediated Clearance Of ImmuneComplexes In Vivo By A Monoclonal Antibody (2.4G2) Directed Against FcReceptors On Murine Leukocytes,” J. Immunol. 133(2):855-862; Unkeless,J. C. (1979) “Characterization of a Monoclonal Antibody Directed AgainstMouse Macrophage and Lymphocyte Fc Receptors,” J. Exper. Med.150:580-596; the sequences for the VH and VL chains of this antibody areavailable from Genbank (ACP40510 and ACP40511, respectively)) wereconstructed, and their respective patterns of glycosylation determined.Table 34 shows the percentages at which the glycosylation species G0F,G1F, G2F, man5, man6, man7, man 8, and man9 were obtained in the Fcvariants of such antibodies (man (mannose), cpx (complexoligosaccharide)).

TABLE 34 Fc Fc Percentage of Total Glycosylation total total Ratio ofVariant Variation G0F G1F G2F man5 man6 man7 man8 man9 man cpx man/cpxAntibody ch4D5 Fc WT WT 43 26 7 11 2 1 1 16 76 0.2  1 P396L 31 30 6 21 76 33 67 0.5  2 Y300L 34 28 5 13 5 3 2 24 67 0.3  3 F243L 7 6 2 31 12 1012 9 75 14 5.2 R292P Y300L P396L  4 F243L 5 9 11 31 8 10 11 7 66 25 2.6R292P Y300L  5 R292P 51 13 1 15 4 3 2 1 24 66 0.4  6 F243L 8 10 5 30 128 7 5 62 23 2.7 R292P P396L  7 F243L 6 28 5 12 16 10 43 33 1.3 R292PP396L  8 F243L 4 11 10 26 16 10 9 5 66 25 2.6 R292P P396L  9 F243L 7 1314 20 16 13 6 3 58 33 1.7 10 F243V 6 12 33 19 14 6 2 41 51 0.8 11 F243R6 4 19 12 24 21 5 62 29 2.1 12 F243C 9 16 29 21 11 5 1 38 54 0.7 13F243F 52 12 2 13 4 4 4 1 27 65 0.4 R292P Y300L 14 F243C 7 12 8 31 13 9 72 63 26 2.4 R292P Y300L 15 F243R 3 3 27 5 29 26 1 61 32 1.9 R292P Y300L16 F243R 3 4 23 4 29 29 2 64 29 2.2 R292P Y300L 17 F243V 5 15 12 22 10 910 6 58 32 1.8 R292P Y300L 18 F243V 5 11 7 30 14 8 9 5 66 24 2.7 R292PY300L 19 L235V 4 9 6 22 12 11 15 12 73 19 3.7 F243L R292P Y300L P396L 20L235V 4 7 3 27 14 11 15 11 78 14 5.4 F243L R292P Y300L P396L 21 F243C 723 23 19 9 6 3 36 53 0.7 R292P 22 F243R 5 5 19 6 25 27 3 61 28 2.2 R292P23 F243V 5 21 29 16 11 6 3 37 55 0.7 R292P 24 F243L 8 19 15 19 8 9 7 447 41 1.2 R292G Y300L 25 F243L 7 17 34 7 19 10 3 39 58 0.7 R292L Y300L26 F243L 7 21 29 12 12 7 3 34 57 0.6 R292S Y300L 27 F243L 6 22 35 8 15 72 33 63 0.5 R292M Y300L 28 F243L 5 8 27 6 23 19 2 50 40 1.3 R292D Y300L29 F243L 7 22 32 10 13 6 2 32 61 0.5 R292Y Y300L 30 F243F 43 22 4 9 4 55 23 69 0.3 R292P P396L 31 F243C 8 21 23 18 9 7 3 37 52 0.7 R292P P396L32 F243R 3 5 22 3 29 28 2 63 31 2.1 R292P P396L 33 F243V 5 19 21 18 5 108 4 45 45 1.0 R292P P396L 34 F243F 36 13 3 13 6 5 9 4 38 51 0.7 R292PY300L P396L 35 F243F 36 12 14 7 5 9 4 39 48 0.8 R292P Y300L P396L 36F243C 8 14 8 23 10 10 10 6 59 29 2.0 R292P Y300L P396L 37 F243R 4 7 26 429 25 58 36 1.6 R292P Y300L P396L 38 F243V 5 12 7 22 11 10 12 9 65 252.6 R292P Y300L P396L 39 F243L 4 19 28 17 12 8 4 41 51 0.8 R292P 40F243L 6 18 14 20 9 9 9 5 52 38 1.4 R292G Y300L P396L 41 F243L 5 19 38 618 9 2 36 61 0.6 R292L Y300L P396L 42 F243L 6 22 26 13 5 12 7 3 39 540.7 R292S Y300L P396L 43 F243L 7 23 34 9 14 7 3 32 63 0.5 R292M Y300LP396L 44 F243L 7 21 28 9 5 14 7 3 38 55 0.7 R292M Y300L P396L 45 F243L 310 30 4 24 19 2 49 43 1.1 R292D Y300L P396L 46 F243L 7 23 25 10 5 11 6 335 55 0.6 R292Y Y300L P396L 47 S239D 26 47 21 6  6 94 0.1 A330L I332EAntibody 2B6 3.5 WT Fc WT 46 28 4 6 4 4 3 2 19 79 0.1 48 F243L 6 16 9 3314 3 4 9 63 30 0.1 R292P V305I 49 F243L 6 6 2 15 13 14 19 18 79 14 0.1R292P Y300L V305I P396L Antibody ch2.4G2 WT Fc WT 57 15 8 7 4 3 2 24 720.1 50 F243L 15 14 15 20 10 10 6 61 30 0.1 R292P V305I 51 F243L 8 5 1014 13 18 25 80 14 0.1 R292P Y300L V305I P396L

Table 35 shows the relative binding (K_(dissociation) wildtype/K_(dissociation) variant) of the CH4D5 IgG Fc of Table 34 to CD16Aand Cd32B. Values greater than 1.0 thus indicate that the IgG variantbound to the Fc receptor with greater affinity than wild-type Fc,whereas values less than 1.0 indicate that the IgG variant bound to theFc receptor with decreased affinity than wild-type Fc.

TABLE 35 Fc Fc Ratio of CD16A(V158) CD16A(F158) CD32B-G2Ag CD32A-G2AgVariant Variation man/cpx (wt/mut) (wt/mut) (wt/mut) (wt/mut) Antibodych4D5 WT Fc WT 0.2 1.0 1.0 1.0 1  1 P396L 0.5 2.2 1.5 2.1 1.8  2 Y300L0.3 1.6 1.2 0.8 1.5  3 F243L 5.2 6.9 4.6 1.2 1.8 R292P Y300L P396L  4F243L 2.6 3.7 2.2 0.5 1.1 R292P Y300L  5 R292P 0.4 1.0 1.0 0.3 0.6  6F243L 2.7 2.3 1.6 0.4 0.8 R292P P396L  7 F243L 1.3 2.3 1.6 0.4 0.8 R292PP396L  8 F243L 2.6 2.3 1.6 0.4 0.8 R292P P396L  9 F243L 1.7 1.1 1.1 0.80.7 10 F243V 0.8 1.0 1.0 0.8 0.7 11 F243R 2.1 0.3 0.3 0.2 0.2 12 F243C0.7 0.7 0.6 0.3 0.3 13 F243F 0.4 2.1 2.0 0.7 1.3 R292P Y300L 14 F243C2.4 2.6 2.1 0.4 0.9 R292P Y300L 15 F243R 1.9 1.1 0.8 0.4 0.9 R292P Y300L16 F243R 2.2 1.1 0.8 0.4 0.9 R292P Y300L 17 F243V 1.8 3.5 2.0 0.5 1.1R292P Y300L 18 F243V 2.7 3.5 2.0 0.5 1.1 R292P Y300L 19 L235V 3.7 5.34.5 0.5 1.3 F243L R292P Y300L P396L 20 L235V 5.4 5.3 4.5 0.5 1.3 F243LR292P Y300L P396L 21 F243C 0.7 2.2 1.1 0.3 0.3 R292P 22 F243R 2.2 n.dn.d n.d n.d R292P 23 F243V 0.7 3.0 1.1 0.2 0.4 R292P 24 F243L 1.2 4.42.5 0.5 1.3 R292G Y300L 25 F243L 0.7 2.6 1.7 0.7 1.6 R292L Y300L 26F243L 0.6 2.9 2.1 0.5 1.4 R292S Y300L 27 F243L 0.5 2.7 1.9 0.7 1.7 R292MY300L 28 F243L 1.3 1.0 0.8 0.4 0.0 R292D Y300L 29 F243L 0.5 1.5 1.4 0.51.1 R292Y Y300L 30 F243F 0.3 1.6 1.8 0.8 1.4 R292P P396L 31 F243C 0.72.3 1.5 0.4 0.7 R292P P396L 32 F243R 2.1 1.2 0.9 0.3 0.6 R292P P396L 33F243V 1.0 2.4 1.8 0.4 0.9 R292P P396L 34 F243F 0.7 4.5 3.4 1.4 2.2 R292PY300L P396L 35 F243F 0.8 4.5 3.4 1.4 2.2 R292P Y300L P396L 36 F243C 2.05.4 3.5 1.1 1.8 R292P Y300L P396L 37 F243R 1.6 1.6 1.5 1.1 1.9 R292PY300L P396L 38 F243V 2.6 6.4 4.1 1.3 2.0 R292P Y300L P396L 39 F243L 0.82.2 1.1 0.3 0.4 R292P 40 F243L 1.4 5.0 3.6 1.1 1.8 R292G Y300L P396L 41F243L 0.6 3.4 2.8 1.2 1.9 R292L Y300L P396L 42 F243L 0.7 2.8 2.2 1.4 2.3R292S Y300L P396L 43 F243L 0.5 3.3 2.3 1.3 2.0 R292M Y300L P396L 44F243L 0.7 3.3 2.3 1.3 2.0 R292M Y300L P396L 45 F243L 1.1 1.4 1.1 0.8 1.5R292D Y300L P396L 46 F243L 0.6 2.1 2.0 1.0 1.7 R292Y Y300L P396L 47S239D 0.1 15.9 11.2 1.5 1.2 A330L I332E Antibody 2B6 3.5 WT Fc WT 0.1 11 1 1 48 F243L 0.1 2.6 1.4 0.2 0.1 R292P V305I 49 F243L 0.1 10.1 8.3 3.21.4 R292P Y300L V305I P396L Antibody ch2.4G2 WT Fc WT 0.1 1 1 1 1 50F243L 0.1 2.6 1.4 0.2 0.1 R292P V305I 51 F243L 0.1 10.1 8.3 3.2 1.4R292P Y300L V305I P396L

The results thus indicate that variations involving positions 243 and292 had pronounced effects on the fucosylation pattern obtained and onFc binding affinity to CD16A, CD32A and CD32B (FIGS. 43-48). In FIGS.43-48, in order to maintain a single y-axis, the percentage of observedman5 or man6 glycosylation patterns (% man56) or of observed man7, man8or man9 glycosylation patterns (% man789) is shown divided by 10. Thus,for example, a reported value of 4.0 in these figures for a % man56glycosylation pattern indicates that 40% of the glycosylation adducts ofthe variant were either man5 or man6. Similarly, the values of totalpercent mannose (% total man) and total complex oligosaccharides (%total cpx) are also shown divided by 10.

FIG. 43 shows the effect of altering the identity of the residuesubstituted at position F243 of a tetra variant (F243X, R292P, Y300L,P396L) on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors. The Figure shows thatmodifications at position 243 alone are sufficient to cause alteredglycosylation and increased affinity to CD16A relative to the wild-type.F243L was particularly capable of increasing affinity to CD16A relativeto wild-type Fc.

FIG. 44 shows the effect of altering the identity of the residuesubstituted at position R292 of a tetra variant (F243L, R292X, Y300L,P396L) on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors. The Figure shows thatmodifications at position 292 alone are sufficient to cause alteredglycosylation and increased affinity to CD16A relative to the wild-type.R292P was particularly capable of increasing affinity to CD16A relativeto wild-type Fc.

FIG. 45 shows the effect of progressive alterations in the identity ofthe residue substituted at position R292, Y300 and P396 of an F243C Fcvariant on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors. The Figure shows that progressivemodifications at these sites synergistically enhanced affinity to CD16Arelative to wild-type Fc.

FIG. 46 shows the effect of progressive alterations in the identity ofthe residue substituted at position R292, Y300 and P396 of an F243L Fcvariant on the observed glycosylation profile of the variant and on therelative binding (K_(dissociation) wild type/K_(dissociation) variant)of the Fc variant to the Fc receptors. The Figure shows that progressivemodifications at these sites synergistically enhanced affinity to CD16Arelative to wild-type Fc, with the tetra variant F243L, R292P, Y300L,P396L exhibiting the largest increase in relative CD16A bindingaffinity.

FIG. 47 shows the effect of progressive alterations in the identity ofthe residue substituted at position R292, Y300 and P396 of an F243Rtetra variant on the observed glycosylation profile of the variant andon the relative binding (K_(dissociation) wild type/K_(dissociation)variant) of the Fc variant to the Fc receptors. The Figure shows thatmodifications at position Y300 or Y396 synergistically enhanced affinityto CD16A of the F243R variant relative to wild-type Fc.

FIG. 48 shows the effect of progressive alterations in the identity ofthe residue substituted at position R292, Y300 and P396 of a tetravariant (F243V, R292X, Y300X, P396X) on the observed glycosylationprofile of the variant and on the relative binding (K_(dissociation)wild type/K_(dissociation) variant) of the Fc variant to the Fcreceptors. The Figure shows that progressive modifications at thesesites synergistically enhanced affinity to CD16A relative to wild-typeFc, with the tetra variant F243V, R292P, Y300L, P396L exhibiting thelargest increase in relative CD16A binding affinity.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A method of attenuating the post-translationalfucosylation of a molecule comprising a modified Fc region, comprising:modifying a human IgG Fc region to comprise an amino acid substitutionrelative to a wild type Fc region to yield a modified Fc region;expressing a candidate molecule comprising said modified Fc region in ahost cell capable of mediating normal glycosylation; determining a ratioof high mannose oligosaccharide glycosylation to complex oligosaccharideglycosylation exhibited by said modified Fc region; identifying saidcandidate molecule comprising said modified Fc region as a suitablemolecule if said modified Fc region exhibits said ratio that is greaterthan about 0.5 and less than about 5.5; and expressing said suitablemolecule possessing said modified Fc region in said host cell.
 2. Themethod of claim 1, comprising the further step of introducing apolynucleotide encoding said modified Fc region into said host cell. 3.The method of claim 1, wherein said amino acid substitution is at one ormore of positions L234, L235, F243, R292, Y300, V305, or P396.
 4. Themethod of claim 3, wherein: (a) said amino acid substitution is F243C,F243L or F243R; (b) said amino acid substitution is R292G, R292L, R292M,R292P, R292S or R292Y; (c) said amino acid substitution is L235V; (d)said amino acid substitution is Y300L; or (e) said amino acidsubstitution is P396L.
 5. The method of claim 1, wherein said modifiedFc region exhibits altered affinity to a CD16A receptor, a CD16Breceptor, a CD32B receptor, or a CD32A receptor.
 6. The method of claim5, wherein said modified Fc region exhibits decreased affinity to aCD32B receptor.
 7. The method of claim 5, wherein said modified Fcregion exhibits increased affinity to a CD16A receptor.
 8. The method ofclaim 1, wherein said modified Fc region is the Fc region of amonoclonal antibody, a chimeric antibody, a humanized antibody, a humanantibody or a single chain antibody.
 9. The method of claim 8, whereinsaid antibody specifically binds CD16A, CD32B, HER2/neu, A33, CD5,CD11c, CD19, CD20, CD22, CD23, CD27, CD40, CD45, CD79a, CD79b, CD103,CTLA4, ErbB1, ErbB3, ErbB4, VEGF receptor, TNF-α receptor, TNF-βreceptor, or TNF-γ receptor.
 10. The method of claim 8, wherein saidantibody specifically binds a cancer antigen associated with a cancer.11. The method of claim 10, wherein said cancer is a breast, ovarian,prostate, cervical, lung or pancreatic cancer.
 12. The method of claim11, wherein said cancer is breast cancer and said antigen is HER2/neu.13. The method of claim 3, wherein said amino acid substitutionscomprise substitutions at positions L234 and L235.
 14. The method ofclaim 13, wherein said amino acid substitutions comprise L234F andL235V.
 15. The method of claim 1, wherein said ratio is: Σ (% Man5+%Man6+% Man7+% Man8+% Man9):Σ (% G0F+% G1F+% G2F).
 16. The method ofclaim 1, wherein said ratio is greater than about 1.0 and less thanabout 5.5.